Encapsulated pre-analytic workflows for flow-through devices, liquid chromatography and mass spectrometric analysis

ABSTRACT

This invention relates to encapsulated workflow reagents comprising an encapsulating material and a workflow reagent encapsulated within the encapsulating material for sample and workflow preparation prior to chromatographic, spectroscopic or other analytical systems, use thereof, and devices comprising the same.

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/367,948, filed Jul. 28, 2016, entitled EncapsulatedPre-Analytic Workflows for Flow-Through Devices, Liquid Chromatographyand Mass Spectrometric Analysis which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Liquid chromatography and mass spectrometry are essential tools inquantifying, analyzing and characterizing a wide variety of molecules.Complex molecules, such as biomolecules, require multiple processingsteps prior to their introduction to flow through devices for analysis.Low molecular weight substances in biological material and bodily fluidsrequire similarly time-consuming, costly and labor-intensivesample-pretreatment steps. Without such preparation, direct injection ofprotein-containing samples would result in improper and incompleteanalysis as well as the accumulation of unwanted species on thechromatographic support materials thereby irreversibly damaging thechromatography column.

Traditional sample preparation often requires multiple steps of reactionand separation. For example, for a particular protein sample to becompletely analyzed for a particular characteristic, it may be necessaryto digest the sample with a particular enzyme or surfactant, or both.The digested peptides may then need to be labeled in some way to allowfor proper detection. In each of these steps, samples are allowed toreact with the particular reagent and then must be separated to removeunwanted materials and reagents prior to chromatographic orspectroscopic analysis.

As an example, a protein digest workflow that uses an affinity capturestep requires multiple reagent addition steps, vortexing, centrifugingand heating steps. In a manual mode this requires the preparation ofreagents, multiple additions and liquid transfer steps, movement ofplates to and from heating blocks, centrifuges, and vortex mixers. Thistype of manual workflow can be labor intensive and can result in highvariability, difficulty transferring and replicating results. The use ofautomation can be used to improve reproducibility and reduce userinvolvement. However, automation requires a significant investment inequipment, supporting infrastructure and requires user generatedscripts. As such the use of automation is often only available in highvolume testing laboratories.

As such, there remains a need for a moderate throughput device thatallows for a singular flow-through workflow device which allows forrobust and simplified workflows.

BRIEF SUMMARY OF THE INVENTION

This invention relates to encapsulated reagents for sample and workflowpreparation, use thereof, and devices comprising the same. Workflowswhich can be used in the materials and methods of the invention include,but are not limited to, reduction or alkylation reagent workflows,protein digest workflows, proteolytic enzyme and protease workflows, andworkflows for the analysis of amino acids, glycoproteins, pesticides andpolar compound analysis.

The use of encapsulated reagents for digestion, hydrolysis anddenaturation (e.g., trypsin, PNGase F, pepsin, IdeS, IdeZ) improvesstability and flowability of these materials. The use of encapsulatedreagents for hydrolytically unstable and reactive molecules (e.g.,WATERS TECHNOLOGIES CORPORATION ACCQ⋅FLUOR™ labeling agent, WATERSTECHNOLOGIES CORPORATION RAPIFLUOR-MS™ tagging reagent, acid-labilesurfactants, acyl chlorides, chloroformate esters, succinimydlcarbonates, esters and isocyanates) allows for improved stability andintroduction at specific points within a workflow. The use ofencapsulated reagents for reactive agents such as reduction agents andalkylating agents (e.g., dithiothreitol (DTT), iodoacetamide,2-mercaptoehtanol, 2-mercaptoethylamine HCl, Tris (2-carboxyethyl)phosphine hydrochloride, 4-vinylpyridine, haloacyl reagents (e.g.acetylchloride), N-ethylmaleimide, bromoalkanes, acrylamide, sodiumborohydride, sodiumcyanoborohydride and other similar reducing agents(e.g. metal hydrides) allows for similarly improved stability andintroduction at specific points within a workflow. Encapsulated reagentscan further be used for stabilizing thermally sensitive standards in theabsence of a singular workflow device. Encapsulated reagents can furtherbe used for stabilizing workflow reagents containing sulfur or otherwisehaving a strong odor. The use of encapsulated reagents for suchcompounds allows for the odor to be masked during sample preparation andanalysis while maintaining the efficacy and efficiency of the reagents.Encapsulated reagents can further be used to minimize harmful exposurein the handling of toxic reagents.

Some reagents, standards, isotopically labeled standards, modifiers, andenzymes are known to be unstable, deactivate or react under ambientconditions, in the presence of co-solvents (such as water), or are ofvariable concentration due to insolubility or adsorption.

Similarly, some enzymes are known to lose some or all activity due to aprocess of autolysis, or self-digestion. The encapsulation of reagents,standards, and enzymes allows for improved stability, transportability,and allows for the introduction of the target group into the workflow ineither a time released or specific manner.

As such, one aspect of the invention provides an encapsulated workflowreagent comprising an encapsulating material and a workflow reagentencapsulated within the encapsulating material.

In certain embodiments of the encapsulated workflow reagent of theinvention, the encapsulating material is attached to a surface of ascaffolding material. In certain embodiments the encapsulating materialis bound to the scaffolding material by coating, by covalent bonding, byionic bonding, through a linker, or a combination thereof.

In other embodiments of the encapsulated workflow reagent of theinvention, the encapsulating material is one or more polymers capable ofproviding a controlled release of the workflow reagents. In particularembodiments, the controlled release is such that the reagent is releasedover a period of time at a controlled rate. In other embodiments, thecontrolled release is such that the reagent is released at a particularpoint of the workflow process.

In still other embodiments of the encapsulated workflow reagent of theinvention, the scaffolding material is a solid, a porous solid, anon-porous solid, a macroporous solid, a mesoporous solid, a microporoussolid, a nanoporous solid, a superficially porous solid, a perfusivesolid, a controlled pore solid, an amorphous solid, a radially alignedporous solid, a non-radially aligned porous solid, a circular orderedporous solid, a crystalline solid, a sintered solid, a liquid, ahydrogel, an aerogel, a xerogel, a cryo-gel, a soft-gel, a gel-likematerial, a wall-anchored monolith, a wall-anchored polymeric highinternal phase material, a particle, a monolith, a membrane, apoly-HIPE, a mesh, a fiber, a screen, an anodized filter, or a frit-likematerial.

In certain embodiments, workflow reagents can be bound or adsorbed to achromatographic material. In such embodiments, the entire workflowreagent including the chromatographic material may be encapsulated bythe encapsulating material. Thus, in certain embodiments, theencapsulated workflow reagent encapsulated within the encapsulatingmaterial is attached to the surface of a chromatographic material. Insuch embodiments, the chromatographic material is a solid, a poroussolid, a non-porous solid, a macroporous solid, a mesoporous solid, amicroporous solid, a nanoporous solid, a superficially porous solid, aperfusive solid, a controlled pore solid, an amorphous solid, a radiallyaligned porous solid, a non-radially aligned porous solid, a circularordered porous solid, a crystalline solid, a sintered solid, a hydrogel,an aerogel, a xerogel, a cryo-gel, a soft-gel, a gel-like material, aparticle material, or a monolith material. In other embodiments, thechromatographic materials including (but not limited to): polymermaterials, silica materials, hybrid organic/inorganic materials,ion-exchange materials, metal impregnated materials, activated carbon,silica, Fluorosil, reversed-phase material, hydrophilic interactionmaterial, hydrophobic interaction materials, desalting materials,restricted access material, or size exclusion material. In certainembodiments in which a workflow reagent and a chromatographic materialare both encapsulated by an encapsulating material, the encapsulatingmaterial may then further be bound to a scaffolding material.

In particular embodiments of the encapsulated workflow reagent of theinvention, the encapsulated workflow reagent material has the formula

[(B)—(Y)_(n))]_(o)-EM  (Formula I)

-   -   EM represents the encapsulating material;    -   B represents a scaffolding material;    -   Y is a linker group between the encapsulating material and a        surface of the scaffolding material;    -   o is an integer greater than or equal to 0; and    -   n is an integer greater than or equal to 1.

In some embodiments having a linker group, the linker group is of theformula represented by Formula II

wherein

n¹ an integer from 0-30;

n² an integer from 0-30;

each occurrence of R¹, R², R³ and R⁴ independently represents hydrogen,fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, aprotected or deprotected alcohol, a zwiterion, or a group Z;

Z represents:

a) a surface attachment group having Formula III:

(B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—   Formula III:

wherein x is an integer from 1-3,

y is an integer from 0-2,

z is an integer from 0-2,

and x+y+z=3

each occurrence of R⁵ and R⁶ independently represents methyl, ethyl,n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted orunsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, aprotected or deprotected alcohol, or a zwiterion group;

B¹ represents —OR⁷, —NR^(7′)R^(7″), —OSO₂CF₃, or —Cl; where each ofR^(7′) R^(7′) and R^(7″) represents hydrogen, methyl, ethyl, n-butyl,iso-butyl, tert-butyl, iso-propyl, thexyl, phenyl, branched alkyl orlower alkyl;

b) an attachment to a surface group through a direct carbon-carbon bondformation or through a heteroatom, ester, ether, thioether, amine,amide, imide, urea, carbonate, carbamate, heterocycle, triazole, orurethane linkage; or

c) an adsorbed, surface group that is not covalently attached to thesurface of the material;

Y′ represents a direct bond; a heteroatom linkage; an ester linkage; anether linkage; an thioether linkage; an amine linkage; an amide linkage;an imide linkage; a urea linkage; a thiourea linkage; a carbonatelinkage; a carbamate linkage; a heterocycle linkage; a triazole linkage;a urethane linkage; a diol linkage; a polyol linkage; an oligomer ofstyrene, ethylene glycol, or propylene glycol; a polymer of styrene,ethylene glycol, or propylene glycol; a carbohydrate group, amulti-antennary carbohydrates, a dendrimer or dendrigraphs, or azwitterion group; and

A represents attachment to the encapsulating material by a ionic group,non-covalently attachment group or by a direct bond including (but notlimited to): a heteroatom linkage; an ester linkage; an ether linkage;an thioether linkage; an amine linkage; an amide linkage; an imidelinkage; a urea linkage; a thiourea linkage; a carbonate linkage; acarbamate linkage; a heterocycle linkage; a triazole linkage; a urethanelinkage; a diol linkage; a polyol linkage; an oligomer of styrene,ethylene glycol, or propylene glycol; a polymer of styrene, ethyleneglycol, or propylene glycol; a carbohydrate group, a multi-antennarycarbohydrates, a dendrimer or dendrigraphs, or a zwitterion group.

In certain embodiments of the encapsulated workflow reagent of theinvention, the workflow reagent is an enzyme, a surfactant, a labelingreagent, a reactive compound, an internal standard, an external standardor a combination thereof.

In some embodiments of the encapsulated workflow reagent of theinvention, the workflow reagent is released over a period of time. Insuch embodiments, the encapsulated workflow reagent of the invention isreleased in proportion to the amount of sample which has been exposed tothe encapsulated workflow reagent of the invention. In certainembodiments, the encapsulated workflow reagent of the invention isreleased immediately upon exposing the encapsulated workflow reagent ofthe invention to a sample.

In one embodiment of the encapsulated workflow reagent of the invention,the primary encapsulation shell further encapsulates one or moreadditional encapsulation shells comprising one or more independentworkflow reagents.

In certain embodiments, the one or more additional encapsulation shellsare in the form of microcapsules separately contained within the primaryencapsulation shell. In certain embodiments, the one or more additionalencapsulation shells are in the form of concentric encapsulation shellssuch that the workflow reagents are sequentially released.

In certain embodiments having one or more additional encapsulationshells such that one or more encapsulation shells—the inner shells—areencapsulated within another encapsulation shell—the outer shell, eachencapsulation shell has an inner surface and an outer surface such thatthe outer surface of one or more inner encapsulation shells is bound tothe inner surface of an outer encapsulation shell. In particularembodiments having one or more additional encapsulation shells, all ofthe encapsulation shells are bound such that the shells remain tetheredtogether upon release of the various workflow reagents and, if present,to the scaffolding material.

In other embodiments, the one or more additional workflow reagents arereleased at the same time. In still other embodiments, the one or moreadditional workflow reagents are released sequentially.

In another aspect, the invention provides a sample preparation devicecomprising an encapsulated workflow reagent according to the invention.In certain embodiments, the device is selected from the group consistingof chromatographic columns, thin layer plates, filtration membranes,sample cleanup devices and microtiter plates; packings for HPLC columns;solid phase extraction (SPE); ion-exchange chromatography; magnetic beadapplications; affinity chromatographic and SPE sorbents; sequesteringreagents; solid supports for combinatorial chemistry; solid supports foroligosaccharide, polypeptides, and/or oligonucleotide synthesis; solidsupported biological assays; capillary biological assay devices for massspectrometry, templates for controlled large pore polymer films;capillary chromatography; electrokinetic pump packing materials; packingmaterials for microfluidic devices; polymer additives; catalysissupports; and packings materials for microchip separation devices.

In still another aspect, the invention provides a method for a methodfor preparing a sample for analysis comprising the steps of:

-   -   providing a sample preparation device comprising an encapsulated        workflow reagent according to the invention;    -   introducing a sample to the sample preparation material; and    -   allowing the sample to remain with the sample preparation        material for sufficient time to release the encapsulated        workflow reagent.

In certain embodiments of the methods of the invention, the methodfurther comprises adding a pore forming agent to induce release of theencapsulated workflow reagent.

In certain other embodiments, the method further comprises contacting anencapsulated workflow reagent with a solvent to induce release of theencapsulated workflow reagent. In particular embodiments, the methodcomprises subjecting an encapsulated workflow reagent with a change inpH to induce release of the encapsulated workflow reagent. In otherparticular embodiments, the method comprises subjecting an encapsulatedworkflow reagent to a change in ionization to induce release of theencapsulated workflow reagent. In other particular embodiments, themethod comprises subjecting an encapsulated workflow reagent to a changein counterion element, counterion charge, or both to induce release ofthe encapsulated workflow reagent. In still other embodiments in which asolvent is used to release the encapsulated workflow reagent, thesolvent is an organic solvent, an aqueous solvent, an aliphatic solvent,an aromatic solvent, an oxygenated solvent, or a halogenated solvent, orwater.

In certain other embodiments, the method further comprises subjecting anencapsulated workflow reagent a change in temperature to induce releaseof the encapsulated workflow reagent.

In certain other embodiments, the method further comprises subjecting anencapsulated workflow reagent a change in ionization to induce releaseof the encapsulated workflow reagent.

In certain other embodiments, the method comprises more than one meansto induce release of the encapsulated workflow reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross sectional depiction of an encapsulated workflowmaterial (5) of the invention in which B is a scaffolding material, Y isa linker group attached to the external surface of the encapsulationshell, (1) represents the internal cavity or void in which a workflowreagent is encapsulated, optionally with other carrier materials orexcipients; (2) represents the internal surface of the encapsulationshell; (3) represents the encapsulation shell; (4) represents theexternal surface of the encapsulation shell; (d1) is the internaldiameter of the encapsulation shell and (d3) is the external diameter ofthe encapsulation shell. In certain embodiments, (1) represents a solid,porous or substantially nonporous material having a surface onto whichthe workflow reagent is bound or adsorbed onto.

FIG. 2 is a cross sectional depiction of an encapsulated workflowmaterial (12) of the invention having an internal encapsulation shellencapsulated within a primary encapsulation shell; in which B is ascaffolding material, Y is a linker group attached to the externalsurface of the primary encapsulation shell, (6) represents the cavity ofthe inner encapsulation shell in which a second workflow reagent isencapsulated; (7) represents the inner encapsulation shell; (8)represents the internal cavity of the primary encapsulation shell inwhich a first workflow reagent and the inner encapsulation shell isencapsulated; (9) represents the internal surface of the primaryencapsulation shell; (10) represents the primary encapsulation shell;(11) represents the external surface of the primary encapsulation shell;(d6) is the internal diameter of the inner encapsulation shell; (d8) isthe internal diameter of the primary encapsulation shell; (d10) is theexternal diameter of the primary encapsulation shell.

FIG. 3 is a cross sectional depiction of an encapsulated workflowmaterial (21) of the invention having an two concentric encapsulationshells (inner and intermediate) encapsulated within a primaryencapsulation shell; in which B is a scaffolding material, Y is a linkergroup attached to the external surface of the primary encapsulationshell, (13) represents the cavity of the inner encapsulation shell inwhich a second workflow reagent is encapsulated; (14) represents theinner encapsulation shell; (15) represents the internal cavity of theintermediate encapsulation shell in which a second workflow reagent andthe inner encapsulation shell is encapsulated; (16) represents theintermediate encapsulation shell; (17) represents the internal cavity ofthe primary encapsulation shell in which a third workflow reagent andthe intermediate encapsulation shell is encapsulated; (18) representsthe internal surface of the primary encapsulation shell; (19) representsthe primary encapsulation shell; (20) represents the external surface ofthe primary encapsulation shell; (d13) is the internal diameter of theinner encapsulation shell; (d15) is the internal diameter of theintermediate encapsulation shell; (d17) is the internal diameter of theprimary encapsulation shell; (d19) is the external diameter of theprimary encapsulation shell.

FIG. 4 is a flowchart depicting an exemplary workflow process foranalysis of proteins.

FIG. 5 is a flowchart depicting an exemplary workflow process foraffinity chromatography.

FIG. 6 is a flowchart depicting an exemplary workflow process for sampledigestion.

FIG. 7 is a flowchart depicting an exemplary workflow process for solidphase extraction (SPE) chromatography.

FIG. 8 is a flowchart depicting an exemplary workflow process foraffinity antibody purification.

FIG. 9 is a flowchart depicting an exemplary workflow process forprotein digestion.

FIG. 10 is a flowchart depicting an exemplary workflow process for solidphase extraction.

FIG. 11A is a cross sectional depiction of an encapsulated workflowmaterial of the invention in which B is a scaffolding material, Y is alinker group attached to the external surface of the encapsulationmaterial, and (1) represents the encapsulating material in which aworkflow reagent is distributed throughout. FIG. 11B is a crosssectional depiction of an encapsulated workflow material of theinvention as in FIG. 11A but with a second encapsulating material layer(2) encapsulating a second workflow reagent coated onto the surface ofthe first encapsulating material (1). The second workflow reagent isdistributed throughout the second encapsulating material (2).

FIG. 12 is a cross sectional depiction of an encapsulated workflowmaterial of the invention in which B is a scaffolding material which mayor may not have a workflow reagent covalently attached or adsorbedthereto and C is an encapsulating material layer coated and, optionally,bonded to the surface of B. Although C may contain a workflow reagentencapsulated therein and evenly distributed throughout, when B containsa workflow reagent covalently attached or adsorbed thereto, C may notcontain a workflow reagent or may optionally include an additionalworkflow reagent.

FIG. 13 is a cross sectional depiction of an encapsulated workflowmaterial of the invention in which B is a scaffolding material which mayor may not have a workflow reagent covalently attached or adsorbedthereto, C is an encapsulating material layer and D is a secondencapsulating material layer. Each encapsulating material layer has aninner and outer surface which may be covalently bonded to the layer ormaterial adjacent to the inner or outer surface of each layer. Layers Cand D may contain one or more workflow reagents encapsulated therein andevenly distributed throughout. In certain embodiments, when B contains aworkflow reagent covalently attached or adsorbed thereto, C or D mayindependently not contain a workflow reagent or may optionally includean additional workflow reagent. In certain embodiments, when C containsa workflow reagent encapsulated therein, D may not contain a workflowreagent or may optionally include an additional workflow reagent.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel workflow materials, e.g., forchromatographic separations, processes and separations devicescontaining the chromatographic material. The present invention will bemore fully illustrated by reference to the definitions set forth below.

“Hybrid”, including “hybrid inorganic/organic material,” includesinorganic-based structures wherein an organic functionality is integralto both the internal or “skeletal” inorganic structure as well as thehybrid material surface. The inorganic portion of the hybrid materialmay be, e.g., alumina, silica, titanium, cerium, or “Hybrid” includesinorganic-based structures wherein an organic functionality is integralto both the internal or “skeletal” inorganic structure as well as thehybrid material surface. The inorganic portion of the hybrid materialmay be, e.g., alumina, silica, titanium, cerium, or zirconium oxides, orceramic material; in an advantageous embodiment, the inorganic portionof the hybrid material is silica. As noted above, exemplary hybridmaterials are shown in U.S. Pat. Nos. 4,017,528, 6,528,167, 6,686,035and 7,175,913 and International Application Publication No.WO2008/103423.

The term “alicyclic group” includes closed ring structures of three ormore carbon atoms. Alicyclic groups include cycloparaffins or naphtheneswhich are saturated cyclic hydrocarbons, cycloolefins, which areunsaturated with two or more double bonds, and cycloacetylenes whichhave a triple bond. They do not include aromatic groups. Examples ofcycloparaffins include cyclopropane, cyclohexane and cyclopentane.Examples of cycloolefins include cyclopentadiene and cyclooctatetraene.Alicyclic groups also include fused ring structures and substitutedalicyclic groups such as alkyl substituted alicyclic groups. In theinstance of the alicyclics such substituents can further comprise alower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a loweralkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, —CF3, —CN, orthe like.

The term “aliphatic group” includes organic compounds characterized bystraight or branched chains, typically having between 1 and 22 carbonatoms. Aliphatic groups include alkyl groups, alkenyl groups and alkynylgroups. In complex structures, the chains can be branched orcross-linked. Alkyl groups include saturated hydrocarbons having one ormore carbon atoms, including straight-chain alkyl groups andbranched-chain alkyl groups. Such hydrocarbon moieties may besubstituted on one or more carbons with, for example, a halogen, ahydroxyl, a thiol, an amino, an alkoxy, an alkylcarboxy, an alkylthio,or a nitro group. Unless the number of carbons is otherwise specified,“lower aliphatic” as used herein means an aliphatic group, as definedabove (e.g., lower alkyl, lower alkenyl, lower alkynyl), but having fromone to six carbon atoms. Representative of such lower aliphatic groups,e.g., lower alkyl groups, are methyl, ethyl, n-propyl, isopropyl,2-chloropropyl, n-butyl, sec-butyl, 2-aminobutyl, isobutyl, tert-butyl,3-thiopentyl and the like. As used herein, the term “nitro” means —NO2;the term “halogen” designates —F, —Cl, —Br or —I; the term “thiol” meansSH; and the term “hydroxyl” means —OH. Thus, the term “alkylamino” asused herein means an alkyl group, as defined above, having an aminogroup attached thereto. Suitable alkylamino groups include groups having1 to about 12 carbon atoms, advantageously from 1 to about 6 carbonatoms. The term “alkylthio” refers to an alkyl group, as defined above,having a sulfhydryl group attached thereto. Suitable alkylthio groupsinclude groups having 1 to about 12 carbon atoms, advantageously from 1to about 6 carbon atoms. The term “alkylcarboxyl” as used herein meansan alkyl group, as defined above, having a carboxyl group attachedthereto. The term “alkoxy” as used herein means an alkyl group, asdefined above, having an oxygen atom attached thereto. Representativealkoxy groups include groups having 1 to about 12 carbon atoms,advantageously 1 to about 6 carbon atoms, e.g., methoxy, ethoxy,propoxy, tert-butoxy and the like. The terms “alkenyl” and “alkynyl”refer to unsaturated aliphatic groups analogous to alkyls, but whichcontain at least one double or triple bond respectively. Suitablealkenyl and alkynyl groups include groups having 2 to about 12 carbonatoms, advantageously from 1 to about 6 carbon atoms.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups and cycloalkylsubstituted alkyl groups. In certain embodiments, a straight chain orbranched chain alkyl has 30 or fewer carbon atoms in its backbone, e.g.,C1-C30 for straight chain or C3-C30 for branched chain. In certainembodiments, a straight chain or branched chain alkyl has 20 or fewercarbon atoms in its backbone, e.g., C1-C20 for straight chain or C3-C20for branched chain, and more advantageously 18 or fewer. Likewise,advantageous cycloalkyls have from 4-10 carbon atoms in their ringstructure and more advantageously have 4-7 carbon atoms in the ringstructure. The term “lower alkyl” refers to alkyl groups having from 1to 6 carbons in the chain and to cycloalkyls having from 3 to 6 carbonsin the ring structure.

Moreover, the term “alkyl” (including “lower alkyl”) as used throughoutthe specification and Claims includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. It willbe understood by those skilled in the art that the moieties substitutedon the hydrocarbon chain can themselves be substituted, if appropriate.Cycloalkyls can be further substituted, e.g., with the substituentsdescribed above. An “aralkyl” moiety is an alkyl substituted with anaryl, e.g., having 1 to 3 separate or fused rings and from 6 to about 18carbon ring atoms, e.g., phenylmethyl (benzyl).

The term “amino,” as used herein, refers to an unsubstituted orsubstituted moiety of the formula —NRaRb, in which Ra and Rb are eachindependently hydrogen, alkyl, aryl, or heterocyclyl, or Ra and Rb,taken together with the nitrogen atom to which they are attached, form acyclic moiety having from 3 to 8 atoms in the ring. Thus, the term“amino” includes cyclic amino moieties such as piperidinyl orpyrrolidinyl groups, unless otherwise stated. An “amino-substitutedamino group” refers to an amino group in which at least one of Ra andRb, is further substituted with an amino group.

The term “aromatic group” includes unsaturated cyclic hydrocarbonscontaining one or more rings. Aromatic groups include 5- and 6-memberedsingle-ring groups which may include from zero to four heteroatoms, forexample, benzene, pyrrole, furan, thiophene, imidazole, oxazole,thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine andpyrimidine and the like. The aromatic ring may be substituted at one ormore ring positions with, for example, a halogen, a lower alkyl, a loweralkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a loweralkylcarboxyl, a nitro, a hydroxyl, —CF3, —CN, or the like.

The term “aryl” includes 5- and 6-membered single-ring aromatic groupsthat may include from zero to four heteroatoms, for example,unsubstituted or substituted benzene, pyrrole, furan, thiophene,imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine,pyridazine and pyrimidine and the like. Aryl groups also includepolycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl andthe like. The aromatic ring can be substituted at one or more ringpositions with such substituents, e.g., as described above for alkylgroups. Suitable aryl groups include unsubstituted and substitutedphenyl groups. The term “aryloxy” as used herein means an aryl group, asdefined above, having an oxygen atom attached thereto. The term“aralkoxy” as used herein means an aralkyl group, as defined above,having an oxygen atom attached thereto. Suitable aralkoxy groups have 1to 3 separate or fused rings and from 6 to about 18 carbon ring atoms,e.g., O-benzyl.

The term “ceramic precursor” “is intended include any compound thatresults in the formation of a ceramic material.

The language “chromatographically-enhancing pore geometry” includes thegeometry of the pore configuration of the presently-disclosed materials,which has been found to enhance the chromatographic separation abilityof the material, e.g., as distinguished from other chromatographic mediain the art. For example, a geometry can be formed, selected orconstructed, and various properties and/or factors can be used todetermine whether the chromatographic separations ability of thematerial has been “enhanced”, e.g., as compared to a geometry known orconventionally used in the art. Examples of these factors include highseparation efficiency, longer column life and high mass transferproperties (as evidenced by, e.g., reduced band spreading and good peakshape.) These properties can be measured or observed usingart-recognized techniques. For example, thechromatographically-enhancing pore geometry of the present porousmaterials is distinguished from the prior art particles by the absenceof “ink bottle” or “shell shaped” pore geometry or morphology, both ofwhich are undesirable because they, e.g., reduce mass transfer rates,leading to lower efficiencies.

Chromatographically-enhancing pore geometry is found in porous materialscontaining only a small population of micropores. Porous materials withsuch a low micropore surface area (MSA) give chromatographicenhancements including high separation efficiency and good mass transferproperties (as evidenced by, e.g., reduced band spreading and good peakshape). Micropore surface area (MSA) is defined as the surface area inpores with diameters less than or equal to 34 Å, determined bymultipoint nitrogen sorption analysis from the adsorption leg of theisotherm using the BJH method. As used herein, the acronyms “MSA” and“MPA” are used interchangeably to denote “micropore surface area”.

The term “functionalizing group” includes organic functional groupswhich impart a certain chromatographic functionality to achromatographic stationary phase.

The term “heterocyclic group” includes closed ring structures in whichone or more of the atoms in the ring is an element other than carbon,for example, nitrogen, sulfur, or oxygen. Heterocyclic groups can besaturated or unsaturated and heterocyclic groups such as pyrrole andfuran can have aromatic character. They include fused ring structuressuch as quinoline and isoquinoline. Other examples of heterocyclicgroups include pyridine and purine. Heterocyclic groups can also besubstituted at one or more constituent atoms with, for example, ahalogen, a lower alkyl, a lower alkenyl, a lower alkoxy, a loweralkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, ahydroxyl, —CF3, —CN, or the like. Suitable heteroaromatic andheteroalicyclic groups generally will have 1 to 3 separate or fusedrings with 3 to about 8 members per ring and one or more N, O or Satoms, e.g. coumarinyl, quinolinyl, pyridyl, pyrazinyl, pyrimidyl,furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl,benzofuranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl,piperidinyl, morpholino and pyrrolidinyl.

The term “metal oxide precursor” is intended include any compound thatcontains a metal and results in the formation of a metal oxide, e.g.,alumina, silica, titanium oxide, zirconium oxide, or cerium oxide.

The term “monolith” is intended to include a collection of individualparticles packed into a bed formation, in which the shape and morphologyof the individual particles are maintained. The particles areadvantageously packed using a material that binds the particlestogether. Any number of binding materials that are well known in the artcan be used such as, for example, linear or cross-linked polymers ofdivinylbenzene, methacrylate, urethanes, alkenes, alkynes, amines,amides, isocyanates, or epoxy groups, as well as condensation reactionsof organoalkoxysilanes, tetraalkoxysilanes, polyorganoalkoxysiloxanes,polyethoxysiloxanes, and ceramic precursors. In certain embodiments, theterm “monolith” also includes hybrid monoliths made by other methods,such as hybrid monoliths detailed in U.S. Pat. No. 7,250,214; hybridmonoliths prepared from the condensation of one or more monomers thatcontain 0-99 mole percent silica (e.g., SiO2); hybrid monoliths preparedfrom coalesced porous inorganic/organic particles; hybrid monoliths thathave a chromatographically-enhancing pore geometry; hybrid monolithsthat do not have a chromatographically-enhancing pore geometry; hybridmonoliths that have ordered pore structure; hybrid monoliths that havenon-periodic pore structure; hybrid monoliths that have non-crystallineor amorphous molecular ordering; hybrid monoliths that have crystallinedomains or regions; hybrid monoliths with a variety of differentmacropore and mesopore properties; and hybrid monoliths in a variety ofdifferent aspect ratios. In certain embodiments, the term “monolith”also includes inorganic monoliths, such as those described in G.Guiochon/J. Chromatogr. A 1168 (2007) 101-168.

The term “nanoparticle” is a microscopic particle/grain or microscopicmember of a powder/nanopowder with at least one dimension less thanabout 100 nm, e.g., a diameter or particle thickness of less than about100 nm (0.1 μm), which may be crystalline or noncrystalline.Nanoparticles have properties different from, and often superior tothose of conventional bulk materials including, for example, greaterstrength, hardness, ductility, sinterability, and greater reactivityamong others. Considerable scientific study continues to be devoted todetermining the properties of nanomaterials, small amounts of which havebeen synthesized (mainly as nano-size powders) by a number of processesincluding colloidal precipitation, mechanical grinding, and gas-phasenucleation and growth. Extensive reviews have documented recentdevelopments in nano-phase materials, and are incorporated herein byreference thereto: Gleiter, H. (1989) “Nano-crystalline materials,”Frog. Mater. Sci. 33:223-315 and Siegel, R. W. (1993) “Synthesis andproperties of nano-phase materials,” Mater. Sci. Eng. A168:189-197. Incertain embodiments, the nanoparticles comprise oxides or nitrides ofthe following: silicon carbide, aluminum, diamond, cerium, carbon black,carbon nanotubes, zirconium, barium, cerium, cobalt, copper, europium,gadolinium, iron, nickel, samarium, silicon, silver, titanium, zinc,boron, and mixtures thereof. In certain embodiments, the nanoparticlesof the present invention are selected from diamonds, zirconium oxide(amorphous, monoclinic, tetragonal and cubic forms), titanium oxide(amorphous, anatase, brookite and rutile forms), aluminum (amorphous,alpha, and gamma forms), and boronitride (cubic form). In particularembodiments, the nanoparticles of the present invention are selectedfrom nano-diamonds, silicon carbide, titanium dioxide (anatase form),cubic-boronitride, and any combination thereof. Moreover, in particularembodiments, the nanoparticles may be crystalline or amorphous. Inparticular embodiments, the nanoparticles are less than or equal to 100nm in diameter, e.g., less than or equal to 50 nm in diameter, e.g.,less than or equal to 20 nm in diameter.

“Surface modifiers” include (typically) organic functional groups whichimpart a certain chromatographic functionality to a chromatographicstationary phase. The porous inorganic/organic hybrid particles possessboth organic groups and silanol groups which may additionally besubstituted or derivatized with a surface modifier.

The language “surface modified” is used herein to describe the compositematerial of the present invention that possess both organic groups andsilanol groups which may additionally be substituted or derivatized witha surface modifier. “Surface modifiers” include (typically) organicfunctional groups which impart a certain chromatographic functionalityto a chromatographic stationary phase. Surface modifiers such asdisclosed herein are attached to the base material, e.g., viaderivatization or coating and later crosslinking, imparting the chemicalcharacter of the surface modifier to the base material. In oneembodiment, the organic groups of a hybrid material, e.g., particle,react to form an organic covalent bond with a surface modifier. Themodifiers can form an organic covalent bond to the material's organicgroup via a number of mechanisms well known in organic and polymerchemistry including but not limited to nucleophilic, electrophilic,cycloaddition, free-radical, carbene, nitrene, and carbocationreactions. Organic covalent bonds are defined to involve the formationof a covalent bond between the common elements of organic chemistryincluding but not limited to hydrogen, boron, carbon, nitrogen, oxygen,silicon, phosphorus, sulfur, and the halogens. In addition,carbon-silicon and carbon-oxygen-silicon bonds are defined as organiccovalent bonds, whereas silicon-oxygen-silicon bonds that are notdefined as organic covalent bonds. A variety of synthetictransformations are well known in the literature, see, e.g., March, J.Advanced Organic Chemistry, 3rd Edition, Wiley, New York, 1985.

The language, “composite material” and the term “composite” are usedinterchangeably herein to describe the engineered materials of theinvention composed of one or more components described herein incombination with dispersed nanoparticles, wherein eachcomponent/nanoparticle remains separate and distinct on a macroscopiclevel within the finished structure. The composite material of thepresent invention is independent of form, and may be monolithic orparticulate in nature. Moreover, a short-hand convention may be used todescribe a composite material containing a dispersed nanoparticle,Np/(A)w(B)x(C)y, and may be understood as follows: the symbolicrepresentation to the left of the slash mark represents the dispersednanoparticle, and the symbolic representations to the right of the slashmark represent the components that comprise the material that thenanoparticle (noted on the left of the slash mark) is dispersed within.In certain embodiments, the composite materials of the present inventionmay be nanocomposites, which are known to include, at least, forexample, nano/nano-type, intra-type, inter-type, and intra/inter-type.(Nanocomposites Science and Technology, edited by P. M. Ajayan, L. S.Schadler, P. V. Braun, Wiley-VCH (Weinheim, Germany), 2003)

As used herein, the terms “aggregates” and “agglomerates” refer toundesired materials generated in the processes of the invention that arelarger than the 90 vol % of the target particle size distribution.Aggregates and/or agglomerates can form from imperfections of the corematerial, improper mixing or dispersion in the process, or excessiveforces during workup. Aggregates and agglomerates can impact theefficiency, permeability, reproducibility and robustness of packed bedswithin chromatographic columns. It is difficult to optimally pack achromatographic column with materials having an elevated amount ofaggregates and agglomerates. Aggregates and agglomerates can break apartwithin a packed bed structure when exposed to high pressures and shears.This can result in a mechanical instability of the packed bed and theresult of a void on the top of the column. This breaking of aggregatesand agglomerates can also result in the generation of fines. Aggregatesand agglomerates can be removed by classification.

As used herein, the term “fines” refers to undesired materials generatedin the processes of the invention that are below the 10 vol % of thetarget particle size distribution. Fines can be formed from reseedingevents or from particle breakage. Resulting fines can be nonporous orfully porous. Often fines are substantially smaller than the 10 vol % ofthe target particle size distribution. Often fines are <1 um in size.Very small fines can cause problems in chromatography in that thepercolate through the packed bed and get stuck in the outlet frit. Thisgenerates increased column pressure. Alternatively fines small enough topercolate through the packed bed and outlet frit can result in problemswith detectors and can contaminate a product. Problems with detectorinclude clogging flow channels, blocking detector windows, and anomalousdetector readings. Such issues can reduce the lifetime of a detector andcan require extensive cleaning protocols. Such issues can also impactthe precision, accuracy, reliability, reproducibility, and robustness ofanalytical data generated. Fines can be removed by classification.

As used herein, the term “substantially nonporous” refers to a materialwhich, although porous, is impermeable or otherwise functions as anon-porous material. Such a substantially nonporous material has a porevolume of less than about 0.10 cc/g.

The term “analyte” is a component, substance or chemical constituentthat is of interest in an analytical procedure. Particular examples mayinclude, but not limited to, drugs, pesticides, polar compounds,herbicides, toxins and environmental pollutants, metal-organiccompounds, biologically active compounds such as metabolites, proteins,peptides, hormones, polynucleotides, vitamins, cofactors, metabolites,lipids and carbohydrates.

The term “sample”, as used herein, refers to a complex fluid mixturecontaining soluble and insoluble components. Particular examplesinclude, but are not limited to, food samples (e.g., milk, a fortifiedfood matrix), biological samples including a sample from human oranimals (e.g., blood, blood plasma, urine, mucosal tissue secretions,tears, semen, and breast milk) and environmental samples (e.g., groundwater, waste waters, soil, and sea, river, pond, or bay water). Thesample may further include macromolecules, e.g., substances, such asbiopolymers, e.g., proteins, e.g., proteolytic proteins or lipophilicproteins, such as receptors and other membrane-bound proteins, andpeptides. The sample may further include one or more lipid, orphospholipid molecules.

The term “matrix” as used herein, refers to the components of a sampleother than the analyte of interest. When the analytes are separated,extracted and analyzed, the matrix may have a substantial interferencewith the analyte during analysis by ESI, or any other technique usingnebulization and ionization of sample components prior to detection suchas APCI and APPI.

The term “matrix interference” as used herein, refers to thosecomponents of the sample that produce a substantial signal enhancementor suppression with the analytes during analysis by mass spectrometry. Asubstantial signal enhancement or suppression that interferes withanalyte quantitation is also termed a substantial interference. Incertain embodiments, the “substantial interference” refers to a matrixeffect that is greater than 20% for targeted analyses and 50% forscreening analyses. Matrix interferences lower than these are consideredacceptable if the majority of analytes in an analysis have matrix effectbelow this threshold.

The term “targeted analysis” as used herein, refers to the analysis of apredetermined set of analytes expected to be present in the samplesbeing analyzed. Targeted analyses typically do not rely on full scanmode data from a MS but from selected ion monitoring of the target ionsof interest. An example of a targeted analysis is quantitatingTacrolimus, an immunosuppressive drug used after organ transplants,given to reduce the risk of rejection. A discrete concentration value isreported for these analyses.

The term “screening analysis” as used herein, refers to the analysis ofa large grouping of analytes that may or may not be present in thesamples being analyzed. An example of a screening analysis isdetermining if pesticides are present in foods. These reports containinformation on the presence of analytes at or above a certain threshold.

The term “matrix effect” or “ME” as used herein, refers to a quantifiedenhancement or suppression of analyte signal. The matrix effect iscalculated for analyte of interest the following formula:

${{Matrix}\mspace{14mu} {Effects}} = {\left( {\frac{\left( {{analyte}\mspace{14mu} {area}\mspace{14mu} {counts}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {presence}\mspace{14mu} {of}\mspace{14mu} {matrix}} \right)}{\left( {{analyte}\mspace{14mu} {area}\mspace{14mu} {counts}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {absence}\mspace{14mu} {of}\mspace{14mu} {matrix}} \right)} - 1} \right) \times 100\%}$

In certain embodiments, the matrix effect is calculated using thefollowing formula:

${{Matrix}\mspace{14mu} {Effects}} = {\left( {\left( \frac{{Response}\mspace{14mu} \left( {{post}\text{-}{spiked}\mspace{14mu} {extracted}\mspace{14mu} {sample}} \right)}{{Response}\mspace{14mu} \left( {{Solvent}\mspace{14mu} {standard}} \right)} \right) - 1} \right) \times 100\%}$

Matrix effects may be caused by, but not limited to, small molecules,proteins, peptides, polymers, surfactants, particulates, cells, lipidsor phospholipids, drug product excipients and salts.

The phrase “solid-phase extraction (SPE) method or protocol” as usedherein, refers to a procedure comprising of the following steps:

-   -   1. Conditioning: the addition of an organic containing wetting        solvent, typically methanol, which is required for the        subsequent use of non-water wettable sorbents like Silica C18.    -   2. Equilibrating the sorbent: the addition of water or buffer to        displace the organic solvent used to condition the sorbent for        use. The removal of the conditioning solution is required for        retention to occur in the following step.    -   3. Loading the sample: the addition of a sample as received or        prepared by dilution and/or, centrifugation or filtration.    -   4. Washing the sorbent: the addition of a solution able to        remove matrix salts, proteins, in particular albumin, and other        polar matrix interferences. The polarity of the wash solution is        such that retention of analytes of interest on the sorbent is        maintained. The wash steps may be repeated multiple times to        remove specific interferences.    -   5. Eluting the analytes: the addition of a solution that elutes        the analytes of interest from the sorbent while minimizing the        co-elution of matrix interferences.

The term “recovery” as used herein, refers to the amount of analyterecovered from the extracted sample. The calculation used for recoveryis shown below:

${Recovery} = {\left( \frac{{Pre}\text{-}{spiked}\mspace{14mu} {sample}\mspace{14mu} {response}}{{Post}\text{-}{spiked}\mspace{14mu} {sample}\mspace{14mu} {response}} \right) \times 100\%}$

The language “biological sample” refers to any solution or extractcontaining a molecule or mixture of molecules that comprises at leastone biomolecule that is subjected to extraction or analysis thatoriginated from a biological source (such as, humans and animals).Biological samples are intended to include crude or purified, e.g.,isolated or commercially obtained, samples. Biological sample may be,but are not limited to, inclusion bodies, biological fluids, biologicaltissues, biological matrices, embedded tissue samples, cells (e.g., oneor more types of cells), and cell culture supernatants. Particularexamples may include blood plasma, urine, cerebrospinal fluid, synovialfluid and other biological fluids, including extracts of tissues, suchas liver tissue, muscle tissue, brain tissue and heart tissue and thelike.

The language “biological matrices” is intended to include anything thata cell contains or makes, e.g., bone, inclusion bodies, bloodcomponents, cell debris, e.g., cell lysates, etc.

The language “biological fluid” as used herein is intended to includefluids that are obtained from a biological source. Exemplary biologicalfluids include, but are not limited to, blood, blood plasma, urine,spinal fluid, mucosal tissue secretions, tears, interstitial fluid,synovial fluid, semen, and breast milk.

The term “chromatographic” process as used herein refers to a processincluding a physical method of separation that distributes components toseparate between two phases, one stationary (stationary phase), theother (the mobile phase) moving in a definite direction.

The term “eluate” as used herein refers to a mobile phase leaving thesample preparation device or column. In certain embodiments, the eluatemay include an analyte of interest or the eluate may not include theanalyte which may be retained by a resin or a matrix of the resin. Insuch instances, the resin or matrix of the resin may be further elutedin a subsequent step to elute the retained analyte of interest.

The term “solid phase extraction (SPE)” refers to a frequently usedchromatographic technique for isolating analytes from the sample forquantitative analysis, especially together with high performance liquidchromatography (HPLC) or gas chromatography (GC) (McDonald and Bouvier,eds. Solid Phase Extraction Applications Guide and Bibliography, sixthedition, Milford, Mass.: Waters (1995)). Solid phase extraction can beadvantageous to separate a component of interest in a complex solutionfrom potentially interfering elements and to concentrate the targetanalytes to the level of sufficient detection and measurement. Forexample, solid phase extraction has been widely utilized in preparingfood or beverage samples, environmental samples and pharmaceuticalagents or metabolites for analysis.

The term “water-wettable” as used herein, describes a material which issolvated, partially or completely, by water. The water-wettable materialengages in energetically favorable or attractive interactions with watermolecules, and thus, maintains its capability for high retention andexcellent recoveries even if the sorbent runs dry, which means there isno need to take extraordinary steps to keep the sorbent beds from dryingout during the critical steps prior to sample loading. Water-wettablematerials are exemplified, but not limited to, those described in U.S.Pat. No. 5,882,521. As observed in the examples of U.S. Pat. No.5,882,521 sorbents that do not engage in favorable energetics orinteractions with water require an organic conditioning followed by anaqueous equilibration in order to maintain retention of analytes ofinterest during sample loading. In certain embodiments, whenwater-wettable material is solvated partially, less than about 1%, lessthan about 3%, less than about 5%, less than about 7%, less than about10%, less than about 15%, less than about 20%, less than about 25%, lessthan about 30%, less than about 35%, less than about 40%, less thanabout 45 less than about 50%,%, less than about 55%, less than about60%, less than about 65%, less than about 70%, less than about 75%, lessthan about 80%, less than about 85%, less than about 90%, less thanabout 95%, or less than about 99% of the material may be solvated or wetby water.

The term “monomer”, as used herein, refers to both a molecule comprisingone or more polymerizable functional groups prior to polymerization, anda repeating unit of a polymer. A polymer can comprise two or moredifferent monomers, in which case it can also be referred to as acopolymer. The “mole percent” of a given monomer which a copolymercomprises is the mole fraction, expressed as a percent, of the monomerof interest relative to the total moles of the various (two or more)monomers which compose the copolymer.

The term “sorption” or “sorbing” describes the ability of a material totake up and hold another material by absorption or adsorption. Withoutadsorption of matrix components and analytes of interest on the surfaceof a sorbent material, retention would not be maintained when the systemis subjected to the flow of certain fluids.

The term “sorbent” refers to a material or molecule capable of sorptionor sorbing. In certain embodiments, the sorbent may adsorb eitheranalytes or other matrix molecules.

As used herein, the term “hydrophobic” refers to a physical or chemicalproperty which repels water or polar molecules. In certain embodiments,the hydrophobic group in a resin makes a substantial interaction oraffinity with hydrophobic analytes or matrix in a sample and adsorbshydrophobic species.

As used herein, the term “hydrophilic” refers to a physical or chemicalproperty which favors water or polar molecules. In certain embodiments,the hydrophilic group in a resin makes a substantial interaction withhydrophilic or polar analytes or matrix in a sample and adsorbshydrophilic species.

The term “phospholipid” refers to a lipid which contains a phosphategroup and one or more of glyceride. The phospholipids are majorcomponents of cell membranes in a form of lipid bilayers. Therefore, thephospholipids are mostly included in biological samples and food andother by-products produced from animal products. In certain embodiments,the phospholipids may cause a matrix effect to substantially disturbanalytical qualification and quantitation because they may interact oradhere to analytes. As such, it is preferred to remove phospholipidprior to analysis.

The terms “analysis” or “analyzing” are used interchangeably and referto any of the various methods of separating, detecting, isolating,purifying, solubilizing, detecting, quantifying and/or characterizingchemical or biological composition. In certain embodiments the analysismay also refer to the various methods of determining the degree ofpurification of a sample. Examples of the various methods include, butare not limited to, solid phase extraction, solid phase microextraction, electrophoresis, mass spectrometry, e.g., MALDI-MS or ESI,liquid chromatography, e.g., high performance, e.g., reverse phase,normal phase, or size exclusion, ion-pair liquid chromatography,liquid-liquid extraction, e.g., accelerated fluid extraction,supercritical fluid extraction, microwave-assisted extraction, membraneextraction, soxhlet extraction, precipitation, clarification,electrochemical detection, staining, elemental analysis, Edmunddegradation, nuclear magnetic resonance, infrared analysis, flowinjection analysis, capillary electrochromatography, ultravioletdetection, and combinations thereof.

The term “mass spectrometric detection” refers to any of the variousmethods of mass spectroscopy. Examples include, but are not limited to,electrospray ionization (ESI), surface desorption ionization, andatmospheric pressure chemical ionization (APCI). In certain embodiments,mass spectrometric detection also includes the use of a tandem massspectrometer, a quadrupole time-of-flight mass spectrometer, or amagnetic sector mass spectrometer.

The language “high-purity analyte/sample” refers to a prepared orextracted analyte which may have reduced contamination and/ornon-diminished chromatographic properties prior to quantitative orqualitative analysis, such as chromatography and mass spectroscopy.

The term “substantially”, as in “substantially higher,” “substantiallygreater” or “substantially improved” as used herein refers to anincrease of an effect of about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% orabout 95% above the original degree of the particular effect.Alternatively, a “substantial” amount may refer to an amount greaterthan about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90% or about 95% of theinitial or total amount. Similarly, “substantially reduced” as usedherein refers to a reduction of an effect of about 10%, about 15%, about20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90% or about 95% above the original degree of the particulareffect.

As used herein the term “substantially free” as in “substantially freeof matrix effects” refers to the reduction or elimination of matrixeffects as a result of a particular workflow reagent such that thematrix effect observed is less than 20% of the effect as compared to theuse of a workflow reagent not encapsulated in accordance with theinvention. In certain embodiments, “substantially free” refers to lessthan 10%, less than 5%, less than 2% less than 1%, less than 0.5% orless than 0.1% of the effect as compared to the use of a workflowreagent not encapsulated in accordance with the invention. In certainother embodiments, “substantially free” refers to less than 1.0%, lessthan 0.7%, less than 0.5%, less than 0.4% less than 0.3%, less than 0.2%or less than 0.1% as compared to the use of a workflow reagent notencapsulated in accordance with the invention. In still otherembodiments, “substantially free” refers to less than 1.0%, less than0.7%, less than 0.5%, less than 0.4% less than 0.3%, less than 0.2% orless than 0.1% as compared to the use of a workflow reagent notencapsulated in accordance with the invention.

Workflow Reagents

In one aspect, the invention comprises encapsulated reagents for use inanalytical workflows and sample preparation. In general, reagents arechosen by the skilled artisan to provide a desired result in theanalytical workflow. The choice of any particular reagent is within theskill of the art.

In certain embodiments, the encapsulated reagent may be an enzyme, asurfactant, a labeling reagent, a reactive compound, or an internal orexternal standard. In certain embodiments, the encapsulated reagent maybe a combination of one or more reagents. In certain other embodiments,the encapsulated reagents may be a combination of one or more types ofreagent. In certain embodiments in which more than one reagent is used,each reagent may be of the same or different types and the reagents maybe contained within the same encapsulation as a mixture or withinseparate encapsulation materials. For example, in FIG. 2, an externalstandard may be encapsulated within an innermost encapsulation materialdesigned to be released at the end of the workflow processing while anenzyme for digestion may be encapsulated within the outer encapsulationmaterial designed to be released at the beginning of the workflowprocessing.

In certain embodiments, the enzyme for analytical workflow is aprotease, cellulase, lipase, amylase, glucoamylase, glucose isomerase,xylanase, phytase, arabinanase, polygalacturonase, hydrolase, chymosin,urease, pectinase, beta-gluconase, ligase, glycosidase, polymerase,phosphatase, kinase, exopeptidase, endopeptidase, aminopeptidase,eramidase or a catalytic protease, such as a serine-protease, threonineprotease, cysteine protease, aspartic protease, glutamic protease orother metallo proteases. In certain embodiments, the enzyme is trypsin,PNGase F, PNGase A, pepsin, chymotrypsin, peptidase, bromelain, papain,IdeS, or IdeZ, elastase, carboxypeptidase A, capthepsin D, capthepsin Eor mixtures thereof. In certain embodiments the enzyme can be insolution or immobilized on a surface of a scaffolding material or aninternal or external surface of an encapsulation shell.

In certain embodiments, the surfactant for analytical workflow is acationic surfactant, a anionic surfactant, a nonionic surfactant, anamphoteric surfactant, a destructible surfactant, or a hydrotrope.

In certain embodiments, the surfactant is selected from the groupconsisting of carboxylates, sulphonates, petroleum sulphonates,alkylbenzenesulphonates, naphthalenesulphonates, olefin sulphonates,alkyl sulphates, sulphates, sulphated natural oils and fats, sulphatedesters, sulphated alkanolamides, and alkylphenols.

In certain embodiments, the surfactant is selected from the groupconsisting of ethoxylated aliphatic alcohol, polyoxyethylenesurfactants, carboxylic esters, .polyethylene glycol esters,anhydrosorbitol ester, glycol esters of fatty acids, carboxylic amides,monoalkanolamine condensates, and polyoxyethylene fatty acid amides.

In certain embodiments, the surfactant is selected from the groupconsisting of quaternary ammonium salts, amines with amide linkages,polyoxyethylene alkyl amines, polyoxyethylene alicyclic amines,4.n,n,n′,n′ tetrakis substituted ethylenediamines, 2-alkyl 1-hydroxethyl2-imidazolines.

In certain embodiments, the surfactant is Lauryl pyridinium,Lauryldimethylbenzyl ammonium, Octylphenoxyethoxyethyl-dimethylbenzylammonium, Cetyltrimethylammonium, Cetylpyridinium, Diethyl heptadecylimidazolinium-1, Diethyl heptadecyl imidazolinium-2, 2M2HT quat-1, 2M2HTquat-2, 2M2HT quat-3, SDS-PAGE, Polysorbates (TWEEN™), Sodium dodecylsulfate (sodium lauryl sulfate), Lauryl dimethyl amine oxide,Cetyltrimethylammonium bromide (CTAB), Polyethoxylated alcohols,Polyoxyethylene sorbitan, Octoxynol (TRITON X100™), N,N-dimethyldodecylamine-N-oxide, Hexadecyltrimethylammonium bromide(HTAB), Polyoxyl 10 lauryl ether, BRIJ 721™, Bile salts (sodiumdeoxycholate, sodium cholate), Polyoxyl castor oil (CREMOPHOR™),Nonylphenol ethoxylate (TERGITOL™), Cyclodextrins, Lecithin,Methylbenzethonium chloride (HYAMINE™),N-coco 3-aminopropionicacid/sodium salt, n-tallow 3-iminodipropionate, disodium salt,N-carboxymethyl n dimethyl n-9 octadecenyl ammonium hydroxide,n-cocoamidethyl n hydroxyethylglycine, sodium salt, 4-BromophenacylBromide, 9-Chloromethylanthracene, N-Chloromethyl-4-nitrophthalimide,N-Chloromethylphthalimide, 3′-Methoxyphenacyl Bromide,O-(4-Nitrobenzyl)-N,N′-diisopropylisourea,1-(4-Nitrobenzyl)-3-p-tolyltriazene, Phenacyl Bromide,3,5-Dinitrobenzoyl Chloride, 2,4-Dinitrofluorobenzene,Nα-(5-Fluoro-2,4-dinitrophenyl)-L-leucinamide,Nα-(5-Fluoro-2,4-dinitrophenyl)-D-leucinamide, Phenyl Isothiocyanate,N-Succinimidyl 4-Nitrophenylacetate,2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl Isothiocyanate,2,3,4,6-Tetra-O-benzoyl-β-D-glucopyranosyl Isothiocyanate,3,5-Dinitrobenzoyl Chloride, 2,4-Dinitrophenylhydrazine Hydrochloride,O-4-Nitrobenzylhydroxylamine Hydrochloride, AABD-SH, Br-Mmc,4-Bromomethyl-6,7-dimethoxycoumarin,3-Bromomethyl-7-methoxy-1,4-benzoxazin-2-one, 9-Chloromethylanthracene,(R)-(−)-DBD-APy, (S)-(+)-DBD-APy, DBD-ED, DBD-PZ, (R)-(−)-NBD-APy,(S)-(+)-NBD-APy, NBD-CO-Hz, NBD-PZ, DBD-COCl, DBD-F, DBD-NCS,(R)-(+)-DBD-Pro-COCl, (S)-(−)-DBD-Pro-COCl, (R)-(−)-DBD-Py-NCS,(S)-(+)-DBD-Py-NCS, 4-(4,5-Diphenyl-1H-imidazol-2-yl)benzoyl ChlorideHydrochloride, NBD-Cl, NBD-, (R)-(+)-NBD-Pro-COCl, (S)-(−)-NBD-Pro-COCl,(R)-(−)-NBD-Py-NCS, (S)-(+)-NBD-Py-NCS, 1,3-Cyclohexanedione, DansylHydrazine, DBD-H, or NBD-H. In certain embodiments the surfactant may bean acid labile surfactant or a destructible surfactant. Examples of suchdestructible surfactants include WATERS TECHNOLOGES COPRPORATIONRAPIGEST™ and those which can be found, without limitation, in U.S. Pat.Nos. 8,580,533; 7,074,936; and 7,229,539; as well as US PatentPublication Nos. US 2006/0057659; US 2006/0094000; and InternationalPatent Publication No. WO 2003/102225, the disclosures of each of whichare incorporated herein by reference.

In certain embodiments, the labeling reagent for analytical workflow isa MS labeling reagent, a fluorescent labeling reagent, a hapten labelingreagent, a photoreactive labeling reagent, a radioisotope labelingreagent, an ultraviolet labeling reagent, a glycan labeling reagent, acovalent isotope-encoding labeling reagent, an amine-modifying labelingreagent, or a functional group-reactive reagent, including, but notlimited to, an amine-reactive reagent. Examples of such labelingreagents can be found, without limitation, in US Patent Publication Nos.US 2014/0179011; US2014/0242709; US2016/0139136; in YING QING YU ET AL.:“A Rapid Sample Preparation Method for Mass SpectrometricCharacterization of N-linked Glycans”, RAPID COMM. MASS SPECTROMETRY,vol. 19, 2005, pages 2331, XP055031335, DOI: doi:10.1002/rcm.2067 and inEuropean Patent Publication No. EP 533200 A1, the disclosures of each ofwhich are incorporated herein by reference.

In certain embodiments, the internal or external standard for analyticalworkflow is a protein standard, a peptide standard, a deuteratedstandard, a radioisotope standard, a fluorescent standard, anultraviolet standard, a glycan standard, a hapten standard, a stableisotope standard capture with anti-peptide antibodies (SISCAPA)standard, or mixtures thereof. Exemplary standards used in quantifying atarget analyte in a sample using LC/MS, can be found in U.S. Pat. Nos.9,274,124; 9,261,506; 9,170,263; 9,163,276; 9,018,580; 8,916,680;8,633,031; 8,580,491; 8,574,860; 8,569,071; 8,568,988; 8,455,202;8,187,893; 8,119,356; 8,097,425; 7,955,810; and 7,807,172, thedisclosures of each of which are incorporated herein by reference.Exemplary standards for other workflows can be found in U.S. Pat. No.8,105,790 or United States Patent Publication No. US 2014/0158881, thedisclosures of each of which are incorporated herein by reference.

In certain embodiments, the reactive compound is a reduction agent or analkylating agent. Particular reactive compounds include, but are notlimited to, dithiothreitol (DTT), iodoacetamide, 2-mercaptoehtanol,2-mercaptoethylamine HCl, Tris (2-carboxyethyl) phosphine hydrochloride,4-vinylpyridine, haloacyl reagents including acetylchloride,N-ethylmaleimide, bromoalkanes, acrylamide, sodium borohydride,sodiumcyanoborohydride and other similar reducing agents such as metalhydrides.

In certain embodiments, the workflow reagent is an agent useful foramino acid analysis.

In certain embodiments, the workflow reagent is useful for analysis andidentification of polar molecules. In particular embodiments, theworkflow reagent is useful for analysis of polar pesticides. Examples ofsuch polar compounds and reagents useful for their analysis andidentification can be found in Journal of Chromatography and SeparationTechniques, Volume 8, Issue, Pages 1000346/1-1000346/6, 2017.

In certain embodiments, workflow reagent may be a reagent having astrong odor. In such embodiments the reagent may bear a sulfur atom. Insuch embodiments, the encapsulation material is capable of reducing,masking or eliminating the odor of the reagent prior to or during theworkflow.

In certain embodiments, workflow reagent may be a toxic or hazardousreagent. In such embodiments, the encapsulation material is capable ofreducing or eliminating the exposure of the skilled artisan performingthe workflow to the toxic or hazardous reagent.

The amounts of each workflow reagent incorporated in any particularseparations device is not particularly limited and is readily determinedprior to any given workflow by one of ordinary skill in the art.

Scaffolding Materials

The invention encompasses encapsulated workflow reagents. In certainembodiments, the encapsulated materials are attached to a secondarysurface or a scaffolding material such as a particle, monolith,membrane, poly-HIPE, mesh, fiber, screen, anodized filters, scaffold, orfrit-like material.

In certain embodiments, the scaffolding material of the inventionincludes, but is not limited to solid, porous solid, non-porous solid,macroporous solid, mesoporous solid, microporous solid, nanoporoussolid, superficially porous solid, perfusive solid, controlled poresolid, amorphous solid, radially aligned porous solid, non-radiallyaligned porous solid, circular ordered porous solid, crystalline solid,amorphous solid, sintered solid, liquid, hydrogel, aerogel, xerogel,cryo-gels, soft-gel, frozen, wax-like, or gel-like material. As thesematerials may be varied in morphology. As such these scaffoldingmaterials prevent loss or leakage of encapsulating material from theseparation device and greatly reduce matrix effects, suppression orenhancement effects due to the encapsulating material in subsequentLC/MS analysis.

Exemplary scaffolding materials include, but are not limited to:wall-anchored monoliths (U.S. Pat. No. 9,289,747 and referencescontained therein), and wall-anchored polymeric high internal phasematerials (Silverstein, M. S. and Cameron, N. R. 2010. PolyHIPEs—PorousPolymers from High Internal Phase Emulsions, Encyclopedia of PolymerScience and Technology; Iacono, M., Connolly, D. and Heise, A. Materials2016, 9, 263; WO 2015/200735; and WO 2015/042592). The internal surfaceof the separation device can include, but are not limited to: steel,stainless steel, titanium, MP35n, PEEK, glass, polymer, polypropylene,polyethylene, copolymers, and Teflon.

In certain embodiments, the internal surface of the separation devicecan be modified before covalent attachment of the scaffolding materialby a number of methods that include, but are not limited to: cleaning,washing with solvents, exposure to bases, exposure to acids, plasmapre-treatment, surface oxidation with plasma, surface treatment withfluoride sources, surface treatment with ozone, surface treatment withnitrogen exposure plasma assisted chemical vapor deposition, oxygenexposure plasma assisted chemical vapor deposition, air exposure plasmaassisted chemical vapor deposition, chemical vapor deposition, molecularvapor deposition, liquid-phase coating approaches, dip-coating,electrochemical coating, initiated polymerization chemical vapordeposition, laser surface texturing, remote plasma sputtering. Whenchemical vapor deposition is employed the surface can be modified by anumber of materials, including but not limited to layers of one or moreof the following: metals (gold, titanium, silver, iron, nickel, copper,molybdenum, chromium) or oxides thereof; silanes or siloxanes, silica,or polymers. Preferred silanes result in surfaces containing silica,organo-silica, or hybrid organic/inorganic materials. Preferred surfacesresult from compositions detailed in U.S. Pat. Nos. 9,248,383;9,211,524; 9,145,481; 9,120,083; 8,791,220; 8,778,453; 8,697,765;8,658,277; 7,919,177; 7,223,473; 6,686,035; and from compositionsdetailed in US 20150212056; 20150136700; 20150133294; 20140329919;20140284259; 20140162298; 20140096596; 20130319086; 20130206665;20130135610; 20130112605; 20120055860 and 20100076103, all of which areincluded herein in their entirety.

The encapsulating material and scaffolding material can display avariety of different groups that are capable of reacting using standardsynthetic protocols to create a covalent linkage. There are a number ofstandard coupling methods known in the literature, including but notlimited to March (Advanced Organic Chemistry, 3rd Edition, Wiley, NewYork, 1985); Odian (The Principles of Polymerization, 2nd Edition,Wiley, New York, 1981); and Bioconjugate Techniques (Hermanson, G. T.,Bioconjugate Techniques; Academic Press: San Diego, 1996)—Repeat.). Theresulting product comprises an encapsulated material attached to atleast one secondary surface. The linker group between the encapsulatingmaterial and the scaffolding material surface is defined in formula Ibelow.

In particular embodiments of the encapsulated workflow reagent of theinvention, the encapsulated workflow reagent material has the formula

[(B)—(Y)_(n))]_(o)-EM  (Formula I)

-   -   EM represents the encapsulating material;    -   B represents the scaffolding material;    -   Y is a linker group between the encapsulating material and the        surface of the scaffolding material;    -   o is an integer greater than or equal to 0; and    -   n is an integer greater than or equal to 1. In certain        embodiments, n is 1, 2, 3, 4, or 5. In certain embodiments, o is        greater than or equal to 1. In other embodiments, o is 1. Such        embodiments are depicted, for example, in FIG. 1.

In some embodiments having a linker group, the linker group is of theformula represented by Formula II

wherein

n¹ an integer from 0-30;

n² an integer from 0-30;

each occurrence of R¹, R², R³ and R⁴ independently represents hydrogen,fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, aprotected or deprotected alcohol, a zwiterion, or a group Z;

Z represents:

a) a surface attachment group having Formula III:

(B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—   Formula III:

wherein x is an integer from 1-3,

y is an integer from 0-2,

z is an integer from 0-2,

and x+y+z=3

each occurrence of R⁵ and R⁶ independently represents methyl, ethyl,n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted orunsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, aprotected or deprotected alcohol, or a zwiterion group;

B¹ represents —OR⁷, —NR^(7′)R^(7″), —OSO₂CF₃, or —Cl; where each ofR^(7′) R^(7′) and R^(7″) represents hydrogen, methyl, ethyl, n-butyl,iso-butyl, tert-butyl, iso-propyl, thexyl, phenyl, branched alkyl orlower alkyl;

b) an attachment to a surface group through a direct carbon-carbon bondformation or through a heteroatom, ester, ether, thioether, amine,amide, imide, urea, carbonate, carbamate, heterocycle, triazole, orurethane linkage; or

c) an adsorbed, surface group that is not covalently attached to thesurface of the material;

Y′ represents a direct bond; a heteroatom linkage; an ester linkage; anether linkage; an thioether linkage; an amine linkage; an amide linkage;an imide linkage; a urea linkage; a thiourea linkage; a carbonatelinkage; a carbamate linkage; a heterocycle linkage; a triazole linkage;a urethane linkage; a diol linkage; a polyol linkage; an oligomer ofstyrene, ethylene glycol, or propylene glycol; a polymer of styrene,ethylene glycol, or propylene glycol; a carbohydrate group, amulti-antennary carbohydrates, a dendrimer or dendrigraphs, or azwitterion group; and

A represents attachment to the encapsulating material by a ionic group,non-covalently attachment group or by a direct bond including (but notlimited to): a heteroatom linkage; an ester linkage; an ether linkage;an thioether linkage; an amine linkage; an amide linkage; an imidelinkage; a urea linkage; a thiourea linkage; a carbonate linkage; acarbamate linkage; a heterocycle linkage; a triazole linkage; a urethanelinkage; a diol linkage; a polyol linkage; an oligomer of styrene,ethylene glycol, or propylene glycol; a polymer of styrene, ethyleneglycol, or propylene glycol; a carbohydrate group, a multi-antennarycarbohydrates, a dendrimer or dendrigraphs, or a zwitterion group.

The scaffolding materials of the invention are capable of forming apacked bed.

In certain embodiments in which the scaffolding materials are particles,the scaffolding materials have an average particle size of the materialis between 0.8-3.0 μm. Specifically, the average particle size of thematerial may be between 1.1-2.9 μm or between 1.3-2.7 μm.

In certain embodiments in which the scaffolding materials are porous,the pores which have an average diameter of about 25-600 Å; about 60-350Å; about 80-300 Å; or about 90-150 Å. In other embodiments in which thescaffolding materials are porous, the scaffolding materials have anaverage pore volume of about 0.11-0.50 cm³/g; about 0.09-0.45 cm³/g; orabout 0.17-0.30 cm³/g. In still other embodiments in which thescaffolding materials are porous, the scaffolding materials have a poresurface area between about 10 m²/g and 400 m²/g.

In some embodiments in which the scaffolding materials are particles,the scaffolding materials of the invention have an average particle sizeof about 0.3-100 μm; about 0.5-20 μm; 0.8-10 μm; or about 1.0-3.5 μm.

Chromatographic Materials

In certain embodiments, workflow reagents can be bound to achromatographic material. In such embodiments, the entire workflowreagent including the chromatographic material may then be encapsulatedby the encapsulating material. Thus, in certain embodiments, theencapsulated workflow reagent encapsulated within the encapsulatingmaterial is attached to the surface of a chromatographic material.

In such embodiments, the chromatographic material is a solid, a poroussolid, a non-porous solid, a macroporous solid, a mesoporous solid, amicroporous solid, a nanoporous solid, a superficially porous solid, aperfusive solid, a controlled pore solid, an amorphous solid, a radiallyaligned porous solid, a non-radially aligned porous solid, a circularordered porous solid, a crystalline solid, a sintered solid, a hydrogel,an aerogel, a xerogel, a cryo-gel, a soft-gel, a gel-like material, awater-wettable material, a particle material, or a monolith material.

In other embodiments, the chromatographic materials including (but notlimited to): polymer materials, silica materials, hybridorganic/inorganic materials, ion-exchange materials, metal impregnatedmaterials, activated carbon, silica, Fluorosil, reversed-phase material,hydrophilic interaction material, hydrophobic interaction materials,desalting materials, restricted access material, or size exclusionmaterial.

In certain embodiments, the chromatographic materials havechromatographically-enhancing pore geometry. In certain embodiments, thechromatographic materials are composite materials. In certainembodiments, the chromatographic materials comprise nanoparticles.

In other embodiments, the chromatographic materials have a surface areaof about 25 to 1100 m²/g; about 80 to 500 m²/g; or about 120 to 330m²/g.

In still other embodiments, the chromatographic materials have a porevolume of about 0.15 to 1.5 cm²/g; or about 0.5 to 1.3 cm²/g.

In other embodiments, the chromatographic materials have a microporesurface area of less than about 110 m²/g; less than about 105 m²/g; lessthan about 80 m²/g; or less than about 50 m²/g.

In some embodiments, the chromatographic materials have an average porediameter of about 20 to 1500 Å; about 50 to 1000 Å; about 100 to 750 Å;or about 110 to 500 Å.

In still embodiments, the chromatographic materials is in the form ofparticles and have an average particle size of about 0.3-100 μm; about0.5-20 μm; 0.8-10 μm; or about 1.0-3.5 μm.

Encapsulation Materials

The concern with encapsulating materials is the release or bleed ofadditional matrix effects, polymers, oligomer or nanoparticles into theLC, MS, LC/MS/MS or LC/MS analysis. For example, insoluble orprecipitated polymeric materials can result in fouling or clogging of anLC, MS, LC/MS/MS or LC/MS system. Such impurities could also co-elutewith a desired target molecules in a chromatographic separation. Moreimportantly the introduction of polymeric, liposomal, or ionizablematerials from the encapsulating material can result in the enhancementor suppression of MS signal. This can impact the sensitivity,quantification, signal-to-noise-ratio, and identification in an MSanalysis.

As such, one aspect of the invention provides for the use ofencapsulating materials that do not release or bleed additional matrixeffects, polymers or nanoparticles into a LC, MS, LC/MS/MS or LC/MSanalysis. This invention allows for the use of encapsulated reagent withimmobilized enzymes in a flow-through device. Additionally, the productof this invention allows for the design of a singular device for complexworkflows that allows for a single sample addition (e.g., whole blood,plasma, serum, urine, tissue) into the device and a single productisolated from the device that is then used in a LC, LC/MS or MSanalysis, in that the specific device is a flow through device that doesnot require the use of centrifugation.

Reducing or eliminating bleed or introduction of matrix effects into theLC, MS, LC/MS/MS or LC/MS analysis can be achieved by a number ofmethods of the present invention. As such, in certain embodiments, theencapsulation materials do not bleed into an LC, MS, LC/MS/MS or LC/MSanalysis. In other embodiments, the encapsulation materials do notionize under MS analysis. In certain embodiments, the encapsulationmaterials are not soluble under conditions used for LC, MS, LC/MS/MS orLC/MS analysis. In certain embodiments, the encapsulation materials arecrosslinked. In certain embodiments, the encapsulation materials areattached to the internal wall of the sample preparation or fluidicdevice.

The encapsulating materials may be made with one or more polymers toprovide a controlled release of the workflow reagents.

In certain embodiments, encapsulating material may be prepared followingthe method of Caruso (Phys. Chem. Chem. Phys., 2011, 13, 4782);Sukhishvili (Chem. Mater., 2006, 18 (2), 328); US20150164805; EP2213280;or Schwendeman (J Control Release. 2014; 196:60). Materials used toprepare encapsulating material include (but are not limited to):emulsifiers, materials with varied melting points, materials withdifferent hydrophilic/lipophilic balances (HLB), phospholipids, fattyacids, plant sterols, sorbitan esters, bees wax, carnauba wax, paraffin,stearates, shellac, cellulose derivatives, maltodextrin, starch, gums,cellulose, Polypyrrole, polycarbonate, cetyltrimethylammonium halides,silanes, diblock copolymers, triblock copolymers such as poly(ethyleneoxide)-blockpoly(propylene oxide)-block-poly(ethylene oxide), named P123(PEO20PPO70PEO20) and F127 (PEO106PPO70-PEO106), alginate, chitosan,xanthan gum, polysaccharide, polysaccharide hydrogel, poly(lysine),poly(acrylic acid), agarose, PEG, poly(hydroxyethylmetacrylate-methylmethacrylate), poly(acrylic acid-co-acrylamide),poly(allylaminehydrochloride), poly(styrenesulfonate sodium salt),poly-(diallyldimethylammonium chloride), poly(ethylene imine),N-hydroxysuccinimide-PEG, maleimide-PEG-conjugated phospholipids,paraffin, cyclodextrin, carboxymethylated polysaccharide,polycaprolactone, humic substances, Span 60, cholesterol, N-trimethylchitosan chloride, poly(methyl methacrylate), poly(2-hydroxyethylmethacrylate), poly(N-isopropylacrylamide),poly(N-isopropylmethacrylamide), poly(N-n-propylacrylamide),carboxymethylcellulose, plastic, gold, molecular weight cut-off filters,hybrid organic/inorganic materials, metal oxides, plastics, silicaincluding SBA-15 (PD 50-89 Å) and MCM-41, ceramics, clays, smecticclays, and niosomes.

In certain embodiments, the encapsulating material contain, or aresubsequently modified to display, a functional group that is capable ofsubsequently reacting with the reactive groups of the co-polymericparticle, using standard synthetic reactions. For example, in certainembodiments, the encapsulating material contains an amino-alkylfunctional group, an ester functional group, an amide functional group,or a carbamate function group which may be reacted with, for example, achloromethyl group of the co-polymeric particle, There are a number ofstandard coupling methods known in the literature, including but notlimited to March (Advanced Organic Chemistry, 3rd Edition, Wiley, NewYork, 1985); Odian (The Principles of Polymerization, 2nd Edition,Wiley, New York, 1981); and Bioconjugate Techniques (Hermanson, G. T.,Bioconjugate Techniques; Academic Press: San Diego, 1996).

In particular embodiments, encapsulating materials containing peripheralamino-alkyl functional groups are then reacted with the scaffoldingmaterial to form a covalent attachment between the encapsulatingmaterial and the co-polymeric particle, using standard syntheticreactions. In certain such embodiments, the linker group between theencapsulating material and the scaffolding material surface can resultfrom the nucleophilic displacement of chlorine to form analkyl-amino-methyl linker (e.g., {(encapsulatingmaterial)-alkyl-NHCH₂}_(n)(co-polymeric particle) where n is ≥1).

Polymeric Materials

Suitable thermoplastic polymers for incorporation as the encapsulationmaterial include, but are not limited to polylactides, polyglycolides,polycaprolactones, polyanhydrides, polyamides, polyurethanes,polyesteramides, polyorthoesters, polydioxanones, polyacetals,polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes,polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates,polyalkylene succinates, poly(malic acid) polymers, polymaleicanhydrides, poly(methylvinyl) ethers, poly(amino acids), chitin,chitosan, and copolymers, terpolymers, or combinations or mixtures ofthe above materials.

Examples of biodegradable polymers and oligomers suitable for use in thecompositions and methods of the present invention include, but are notlimited to: poly(lactide)s; poly(glycolide)s;poly(lactide-co-glycolide)s; poly(lactic acid)s; poly(glycolic acid)s;and poly(lactic acid-co-glycolic acid)s; poly(caprolactone)s; poly(malicacid)s; polyamides; polyanhydrides; polyamino acids; polyorthoesters;polyetheresters; polycyanoacrylates; polyphosphazines;polyphosphoesters; polyesteramides; polydioxanones; polyacetals;polyketals; polycarbonates; polyorthocarbonates; degradablepolyurethanes; polyhydroxybutyrates; polyhydroxyvalerates; polyalkyleneoxalates; polyalkylene succinates; chitins; chitosans; oxidizedcelluloses; and copolymers, terpolymers, blends, combinations ormixtures of any of the above materials.

As used herein, “hydrophobic” refers to a polymer that is substantiallynot soluble in water. As used herein, “hydrophilic” refers to a polymerthat may be water-soluble or to a polymer having affinity for absorbingwater, but typically not when covalently linked to the hydrophobiccomponent as a co-polymer, and which attracts water into the device.

The term “log P” as used herein, refers to the octanol:water partitioncoefficient. The formula for the partition coefficient is Log P=[analyteconcentration in octanol]/[analyte concentration in water]. The log Pvalue is used as a measure of an analytes hydrophobicity orhydrophobicity where values of less than 1 indicate that the analyteconcentration if higher in water than in octanol. As such the analytewould be considered a more hydrophilic or polar analyte. Log P valuesgreater than 1 indicate that an analyte is more hydrophobic ornon-polar. The higher the log P value correlates with a higherhydrophobicity relative to lower values on the same scale. The lower thelog P value relative to 1 indicates that the analyte is more hydrophilicor more polar than others of higher value.

Hydrophilic polymers suitable for use herein can be obtained fromvarious commercial, natural or synthetic sources well known in the art.Suitable hydrophilic polymers include, but are not limited to:polyanions including anionic polysaccharides such as alginate; agarose;heparin; polyacrylic acid salts; polymethacrylic acid salts; ethylenemaleic anhydride copolymer (half ester); carboxymethyl amylose;carboxymethyl cellulose; carboxymethyl dextran; carboxymethyl starch;carboxymethyl chitin/chitosan; carboxy cellulose;2,3-dicarboxycellulose; tricarboxycellulose; carboxy gum arabic; carboxycarrageenan; carboxy pectin; carboxy tragacanth gum; carboxy xanthangum; carboxy guar gum; carboxy starch; pentosan polysulfate; curdlan;inositol hexasulfate; beta.-cyclodextrin sulfate; hyaluronic acid;chondroitin-6-sulfate; dermatan sulfate; dextran sulfate; heparinsulfate; carrageenan; polygalacturonate; polyphosphate;polyaldehydo-carbonic acid; poly-1-hydroxy-1-sulfonate-propen-2;copolystyrene maleic acid; mesoglycan; sulfopropylated polyvinylalcohols; cellulose sulfate; protamine sulfate; phospho guar gum;polyglutamic acid; polyaspartic acid; polyamino acids; and anyderivatives or combinations thereof. One skilled in the art willappreciate other hydrophilic polymers that are also within the scope ofthe present invention.

Various water-soluble polymers suitable for use herein include, but arenot limited to: poly (alkyleneglycol), polyethylene glycol (“PEG”);propylene glycol; ethylene glycol/propylene glycol copolymers;carboxylmethylcellulose; dextran; polyvinyl alcohol (“PVOH”); polyvinylpyrolidone; poly (alkyleneamine)s; poly (alkyleneoxide)s;poly-1,3-dioxolane; poly-1,3,6-trioxane; ethylene/maleic anhydridecopolymers; polyaminoacids; poly (n-vinyl pyrolidone); polypropyleneoxide/ethylene oxide copolymers; polyoxyethylated polyols; polyvinylalcohol succinate; glycerine; ethylene oxides; propylene oxides;poloxamers; alkoxylated copolymers; water soluble polyanions; and anyderivatives or combinations thereof. In addition, the water-solublepolymer may be of any suitable molecular weight, and may be branched orunbranched.

In the practice of the invention, the hydrophobic polymer component isco-polymerized with a hydrophilic polymer, or monomers, to yield apolymer system, most preferably a block copolymer, or blended with ahydrophilic polymer to yield a blended polymer system. These resultantpolymer systems are characterized as having a small amount ofhydrophilic character, but they will not form a hydrogel followingimmersion in an aqueous system. For example, preferred polymer systemsfor use in the compositions of the present invention may contain awater-soluble polymer such as polyethylene glycol (PEG) in amountstypically up to 25 to 30 wt %, not imparting the hydrogel propertiescited by Churchill but producing devices that exhibit monophasic orzero-order or near zero-order release kinetics. If a PEG is used in thesystem, the preferred molecular weight may be between about 700 Da andabout 500 kDa. Other particularly preferred hydrophilic polymers for usein the polymer systems of the invention include polyvinyl pyrolidone,polyvinyl alcohols, poly (alkyleneamine)s and poly (alkyleneoxide)s.

As used herein, “polymer” and “polymer system” include copolymers andblends unless otherwise expressly defined. The polymer systems can beproduced using standard copolymerization techniques, such as graftcopolymerization, polycondensation and polyaddition, optionally with anappropriate catalyst. These techniques can be carried out inconventional manner well known in the polymer art as regards to time andtemperature. Alternatively, the polymer systems can be produced usingstandard blending techniques of polymers or blending of copolymers,again carried out in conventional manner well known in the polymer artas regards to time and temperature.

Depending on the desired softness and flexibility of the encapsulationmaterial, the rate and extent of reagent release, rate of degradation,and the like, the amount and type of polymer can be varied to producethe desired result. For example, for a relatively soft and flexiblepolymer system, copolymers with a low T_(g) can be used, primarily thelactide/caprolactone copolymers. The ratio of glycolide to lactide or tocaprolactone can also be varied to effect water diffusibility, whichincreases with an increasing amount of the more hydrophilic monomer. Thehydrophilic character of these monomers increases in the series ascaprolactone<lactide<glycolide.

Encapsulation Shells and Impregnated Layers

In certain embodiments, the polymers form an encapsulation shell whichmay be rendered porous under certain conditions and over time, therebycontrolling the release. The pores can be formed by a swelling of thepolymer shell or by a dissolution or degradation of the shell. Incertain embodiments, the encapsulation materials comprise one or morereagents. In certain other embodiments, the encapsulations materialscomprise one or more reagents and one or more additional encapsulationmaterials within the shell thereby forming a series of shells whichrelease the reagent at different times. In such instances, the materialscan take the form of a series of concentric encapsulation shells whichallow for the sequential release of reagents. In such instances, it ispreferred that the encapsulation shell/sphere is incapable of passingthrough the pores of the shell in which it is encapsulated but that thereagent encapsulated therein is released after a desired time. In othersuch instances, the materials can be formed in microcapsules containedwithin the primary/outermost encapsulation shell. In such instances,different microcapsules may release their reagent at the same timethereby allowing reagents which must remain separate until reaction todo so. In such instances, it is preferred that encapsulatedmicrocapsules remain within the primary/outermost encapsulation shellwithout passing through the pores therein.

In certain embodiments having one or more additional encapsulationshells such that one or more encapsulation shells—inner shells—areencapsulated within another encapsulation shell—outer shell, eachencapsulation shell has an inner surface and outer surface such that theouter surface of one or more inner encapsulation shells is bound to theinner surface of an outer encapsulation shell. In particular embodimentshaving one or more additional encapsulation shells, all of theencapsulation shells are bound such that the shells remain tetheredtogether upon release of the various workflow reagents and, if present,the scaffolding material.

The mass, volume and thickness of the polymers in each encapsulationshell/sphere can also be varied to adjust the release rate of theincorporated reagent.

The use of the term shell/sphere, as used herein, is not limiting as tothe shape of the encapsulation material. Although the shape of thematerial is generally spherical, it is possible to prepare and utilizeconical shells, tubular shells, oblong shells, cylindrical rods, and thelike.

In certain embodiments the material may be amorphous or irregularlyshaped. In certain other embodiments the encapsulation material may becoated or bonded to the surface of a scaffolding material or achromatographic material. In such embodiments, the encapsulationmaterial may take the shape of the material to which it is bonded. Incertain embodiments in which the chromatographic material or scaffoldingmaterial is porous, the encapsulation may or may not penetrate the poresof the underlying material.

In certain other embodiments, the polymers may be impregnated with theworkflow reagent and coated onto the surface of a scaffolding material.In such embodiments, the workflow material is blended or mixed with thepolymers such that the reagent becomes embedded, encapsulated orimpregnated into the polymer matrix. In such embodiments, theencapsulation material does not form a discreet encapsulation shell but,instead, the encapsulation material containing the workflow reagent maybe coated onto a scaffolding material as a layer and, optionally,covalently or ionically bonded thereto. In certain embodiments in whichmore than one workflow reagent is used, the different workflow reagentsare impregnated into the same encapsulation material layer. In otherembodiments, in which more than one workflow reagent is used, thedifferent workflow reagents are impregnated into different encapsulationmaterial layers which are coated sequentially such that the innermostlayer—the layer coating the scaffolding material—is released last whilethe outermost layer—the layer having no subsequent layer coated orbonded thereto—is released first. Such embodiments are depicted, forexample, in FIGS. 11-13.

In certain embodiments, the encapsulation material may be a wax,hydrogel, a silicone rubber, or a trehalose glass. The use of hydrogels,silicone rubbers and trehalose glasses is particularly suited for theimpregnation of workflow reagents into the polymer material though anysuitable polymer may be used in such embodiments.

In still other embodiments, the workflow reagent can be loaded into thepores of a porous material, for example, a porous scaffolding materialor a porous chromatographic material. In such embodiments, once theworkflow reagent is loaded into the pores of the porous material, theporous material can be coated with one or more polymeric encapsulationmaterials as described herein. In such embodiments, the polymericencapsulation material may further include one or more additionalworkflow reagents which are impregnated into the encapsulation materialas discussed above.

Inducing Release

The encapsulated workflow reagents may be released from theencapsulating materials by any number of means as may be known to one ofordinary skill in the art. In certain embodiments of the invention, suchrelease can be induced by contacting the encapsulating material with apore-forming agent. In other embodiments of the invention, the releasecan be induced by a physical change or a chemical change. For example,and without limitation, the release can be induced by changes intemperature, pH, ionic charge, counterion charge, or counterion atom.Similarly, the release can be induced by contacting the encapsulatingmaterial with a solvent including, but not limited to, an organicsolvent, an aqueous solvent, an aliphatic solvent, an aromatic solvent,an oxygenated solvent, or a halogenated solvent, or water. Depending onthe particular workflow and encapsulating materials, the release may beinduced by a combination of means for release.

In general, the release rate for a reagent will be determined by theskilled artisan based on the particular workflow being utilized. Incertain embodiments, the desired release rate is immediate whereas inother embodiments the release rate is controlled so that the reagent isreleased over a period of time. In certain embodiments, the reagent isreleased over the course of the workflow such that about 100% of thereagent is released by the time that about 100% of the sample has beenintroduced. In other embodiments, the reagent is released over thecourse of the workflow such that about 100% of the reagent is releasedby the time that about 90% of the sample has been introduced; by thetime that about 80% of the sample has been introduced; by the time thatabout 75% of the sample has been introduced; by the time that about 50%of the sample has been introduced; or by the time that about 25% of thesample has been introduced. In other embodiments, when multiple reagentsare encapsulated, the release of each reagent is optimized for theparticular workflow being performed.

Pore-Forming Agents

Other additives can be used to advantage in further controlling thedesired release rate of a reagent for a particular workflow protocol.For example, if the thermoplastic polymer liquid composition is tooimpervious to water, a pore-forming agent can be added to generateadditional pores in the matrix. Any compatible water-soluble materialcan be used as the pore-forming agent. These agents can be eithersoluble in the liquid composition or simply dispersed within it. Theyare capable of dissolving, diffusing or dispersing out of both thecoagulating polymer matrix and the formed polymer system whereupon poresand microporous channels are generated in the matrix and system. Theamount of pore-forming agent (and size of dispersed particles of suchpore-forming agent, if appropriate) within the composition will directlyaffect the size and number of the pores in the polymer system.

Other factors can also influence the size and/or diameter of the poresformed in the polymer system. For example, the amount of organicsolvent, and the rate at which the polymer system solidifies, can allaffect the porosity of the polymer system. Although a generallymicroporous matrix without a resolved core and skin can be producedaccording to the invention, typically, without an additionalpore-forming agent a polymer system formed from the liquid compositionis composed of a surface skin and inner core. The surface skin istypically less porous, and even relatively nonporous, when compared tothe inner core. The inner core can contain pores with a diameter ofabout 10-1000 um. With additional pore-forming agent, the pore sizes ofthe core and skin become substantially uniform such that they both havepores in the range of 10 to 1000 um.

The concentration of pore-forming agent relative to thermoplasticpolymer in the composition will vary according to the degree ofpore-formation desired. Generally, this concentration will range fromabout 0.01 to 1 gram of pore-forming agent per gram of polymer. If theagent is soluble in the liquid composition, then the mixing ordistribution of the agent in the liquid composition and the aggregationwhen the thermoplastic coagulates will determine the size of theresultant pores as the agent dissolves out of the polymer matrix.

Pore-forming agents include any pharmaceutically acceptable organic orinorganic substance that is substantially miscible in water and bodyfluids and will dissipate from the forming and formed matrix intoaqueous medium or body fluids or water-immiscible substances thatrapidly degrade to water-soluble substances. The pore-forming agent maybe soluble or insoluble in the polymer liquid composition of theinvention. In the liquid composition of the invention, it is furtherpreferred that the pore-forming agent is miscible or dispersible in theorganic solvent to form a uniform mixture. Suitable pore-forming agentsinclude, for example, sugars such as sucrose and dextrose, salts such assodium chloride and sodium carbonate, and polymers such ashydroxylpropylcellulose, carboxymethylcellulose, polyethylene glycol,and polyvinylpyrrolidone. The size and extent of the pores can be variedover a wide range by changing the molecular weight and percentage ofpore-forming agent incorporated into the polymer system.

Other excipient materials can be added to the devices to alter porosity,for example, materials like sucrose, dextrose, sodium chloride,sorbitol, lactose, polyethylene glycol, mannitol, fructose, polyvinylpyrrolidone or appropriate combinations thereof. Additionally, theactive agents may be dispersed with oils (e.g., sesame oil, corn oil,vegetable), or a mixture thereof with a phospholipid (e.g., lecitin), ormedium chain fatty acid triglycerides (e.g., Miglyol 812) to provide anoily suspension.

Devices and Methods

Another aspect provides a variety of separations and analytic deviceshaving a stationary phase comprising the materials as described herein.The separations devices include, e.g., chromatographic columns, thinlayer plates, filtration membranes, sample cleanup devices andmicrotiter plates; packings for HPLC columns; solid phase extraction(SPE); ion-exchange chromatography; magnetic bead applications; affinitychromatographic and SPE sorbents; sequestering reagents; solid supportsfor combinatorial chemistry; solid supports for oligosaccharide,polypeptides, and/or oligonucleotide synthesis; solid supportedbiological assays; capillary biological assay devices for massspectrometry, templates for controlled large pore polymer films;capillary chromatography; electrokinetic pump packing materials; packingmaterials for microfluidic devices; polymer additives; catalysissupports; and packings materials for microchip separation devices.Similarly, materials of the invention can be packed into preparatory,microbore, capillary, and microfluidic devices.

In certain embodiments, the encapsulated materials of the invention areused in a separation device, a sample preparation device, or a fluidicdevice. As such, one aspect of the invention encompasses a separationdevice, a sample preparation device, or a fluidic device comprising anencapsulated material of the invention.

In certain embodiments, the encapsulation materials are prevented frompermeation or bleeding from the separation device by the use ofmolecular weight cut-off filters, ultra-filtration, membranes, or frits.

In specific embodiments, the sample preparation or a fluidic deviceallows for removal through adsorption of the undesired matrix effectsintroduced from the encapsulating materials and the elution of thedesired target analytes or precursors from the flow-through device withor without active gating or valving.

In other embodiments, the sample preparation or a fluidic device allowsfor adsorption of desired target analytes or precursors on achromatographic material, and the elution of undesired matrix effectsintroduced from the encapsulating materials by elution from theflow-through device with or without active gating or valving.

In other embodiments, the sample preparation or a fluidic device allowsfor a chromatographic separation of the desired target analytes orprecursors from the undesired matrix effects introduced from theencapsulating materials, with or without the use of gating or valving inthe flow through device. In this separation the desired target analytesor precursors elute before or after the undesired matrix effects.

In other embodiments, the devices comprise chromatographic materialsincluding (but not limited to): polymer materials, silica materials,hybrid organic/inorganic materials, ion-exchange materials, metalimpregnated materials, activated carbon, silica, Fluorosil,reversed-phase material, hydrophilic interaction material, hydrophobicinteraction materials, desalting materials, restricted access material,or size exclusion material.

In certain embodiments, the sample preparation or a fluidic deviceincludes the use of Enzyme immobilization (Chem. Soc. Rev., 2009, 38,453-468)

In one embodiment, the device may further include a housing. Thehousing, as used herein, may have a chamber for accepting samples andthe encapsulated material. The housing in the present invention may alsobe used for a chromatographic column or device without limitation.

In certain exemplary embodiments, the housing of the devices may be asingle chamber or the housing has a plurality of chambers. In addition,the housing may also be constructed and arranged to present the chambersin a plate-like format as described in US Pub. No.: US 2013/0053588 A1and U.S. Pat. No. 6,723,236, each of which is incorporated herein byreference in its entirety. In yet certain exemplary embodiments, thehousing may be, but are not limited to, a syringe, a cartridge, acolumn, a multiple chamber, a 8-well rack, a 8-well strip, a 96-wellplate, 96-well micro-elution plate, micro-elution tip, a96-micro-elution tip array, an 8-tip micro-elution strip, a singlemicro-elution pipet tip, a spin tube or a spin container. However, anynumber of chambers or shape of the chambers may be obtained based upon auser's requirements.

In certain embodiments, a solution received in the housing may flow intothe device spontaneously, for example, by capillary action.Alternatively, the flow may be generated through the device by externalforces, such as gravity, a vacuum chamber, reservoir or a structure toallow for use in a centrifuge, or with a vacuum or pressure source,external pressure and the like, without limitation.

In certain exemplary embodiments, the housing may be configured to haveat least one cap or lid for closing the housing after the sample isreceived. Also, the cap may provide a pressure source for efficientextraction process. In certain yet exemplary embodiments, the cap may beassociated with a vacuum chamber, reservoir or a structure to allow foruse in a centrifuge, or with a vacuum or pressure source to apply vacuumor suction to facilitate flow out of the solution or liquid throughoutthe device to the outlet. In particular, a vacuum chamber, reservoir ora structure to allow for use in a centrifuge or with a vacuum orpressure source may also be associated with other component of thehousing without limitation, as known in the art. In particularembodiments, the housing may be configured to have a hydropobic frit toallow for retention of aqueous solutions.

In certain exemplary embodiments, the housing may be formed of asubstantially rigid material which withstands sufficient mechanicalstrength applied during the extraction. Exemplary materials may be, butnot limited to, a glass, a metal, a plastic and the like. In anexemplary embodiment, the molded plastic such polyether ether ketone(PEEK), polycarbonate (PC), and the like may be used without limitation,as known in the art.

In certain exemplary embodiments, a capacity of the housing may bevarious according to the volume of the sorbents, samples and the likebased on a user's requirements. Exemplary housings of the invention mayhave a capacity of about 1 mL, about 2 mL, about 3 mL, about 4 mL, about5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about15 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, or about 100mL, without limitation.

In one embodiment, the device may further include an inlet or an outlet.

In one embodiment, the device of the invention may include one or moreof frits or filters. For instance, a frit or a filter may be disposed atthe bottom of the sorbents or a frit or a filter may be disposed on thetop of the sorbents. The frits or filters, as used herein, may maintainstabilization or equilibration the sorbents until sufficient flow isgenerated through the device. In other words, the frits or filters mayprevent the sorbents from being penetrated by the sample solution untila sufficient external force is applied. In certain exemplaryembodiments, a porous frit may be used. Exemplary frits may be, but notlimited to, a hydrophobic frit material such as Teflon, which may be ina form of membrane, filter, screen or monolithic structure orcombination thereof. In yet certain exemplary embodiments, fits may notbe disposed in the sorbents. In other certain exemplary embodiments,frits may be disposed between layers of sorbents, without limitations tonumber or orders.

In certain aspects, the invention provides a method for preparing asample for chromatographic analysis comprising the steps of:

providing a sample preparation material comprising an encapsulatedworkflow reagent;

introducing a sample to the sample preparation material; and

allowing the sample to remain with the sample preparation material forsufficient time to release the encapsulated workflow reagent.

In certain embodiments, the release of the encapsulated workflow isinduced by introduction of a pore forming agent or a solvent.

In certain other embodiments, the method further comprises the steps ofseparating the sample from the sample preparation material and analyzingthe sample on a chromatographic device.

In certain embodiments, the encapsulation material and/or the workflowreagents are chosen so as to minimize matrix interference or signalsuppression. The term “matrix interference” as used herein, refers tothose components of the sample that produce a substantial signalenhancement or suppression with the analytes during analysis by massspectrometry. A substantial signal enhancement or suppression thatinterferes with analyte quantitation is also termed a substantialinterference. In certain embodiments, the “substantial interference”refers to a matrix effect that is greater than 20% for targeted analysesand 50% for screening analyses. Matrix interferences lower than theseare considered acceptable if the majority of analytes in an analysishave matrix effect below this threshold.

One method for determining potential matrix effects utilizes post-columninfusion of eluates derived using blank (unspiked) plasma. A cleanstandard (1), containing the analytes of interest, is injected on thecolumn. A tee of the infused eluates combines with the fluid from thecolumn outlet. The combined flow is then directed into the MS foranalysis.

The MS detector analyzes each analyte, as it elutes from the column, inthe presence of the blank plasma eluates from the various samplepreparation methods to be compared. This method shows matrix effectsdifferences attributed to matrix substances remaining in the eluate. TheMS response for each of the analytes while infusing matrix-free elutionsolutions is compared to the MS response for each of the analytes whileinfusing eluates from processing blank plasma by the cited method. Thisapproach to accessing matrix effects in known to those familiar with theart. Examples of a reference for this approach is C. Polson et al./J.Chromatogr. B 785 (2003) 263-275.

Further methods for avoiding matrix interference can be found, forexample, in International Patent Application Serial No. PCT/US15/65993.International Patent Application Serial No. PCT/US15/65993 also providesexemplary chromatographic devices, columns, and packing materials whichmay be adapted to include the encapsulated workflow reagents of theinvention. The disclosure of International Patent Application Serial No.PCT/US15/65993 is incorporated herein by reference.

EXAMPLES

Characterization

Those skilled in the art will recognize that equivalents of thefollowing instruments and suppliers exist and, as such, the instrumentslisted below are not to be construed as limiting.

The % C values were measured by combustion analysis (CE-440 ElementalAnalyzer; Exeter Analytical Inc., North Chelmsford, Mass.) or byCoulometric Carbon Analyzer (modules CM5300, CM5014, UIC Inc., Joliet,Ill.). The specific surface areas (SSA), specific pore volumes (SPV) andthe average pore diameters (APD) of these materials were measured usingthe multi-point N₂ sorption method (Micromeritics ASAP 2400;Micromeritics Instruments Inc., Norcross, Ga.). The SSA was calculatedusing the BET method, the SPV was the single point value determined forP/P₀>0.98 and the APD was calculated from the desorption leg of theisotherm using the BJH method. The micropore surface area (MSA) wasdetermined as the cumulative adsorption pore diameter data for pores <34Å subtracted from the specific surface area (SSA). The median mesoporediameter (MPD) and mesopore pore volume (MPV) were measured by mercuryporosimetry (Micromeritics AutoPore IV, Micromeritics, Norcross, Ga.).Skeletal densities were measured using a Micromeritics AccuPyc 1330Helium Pycnometer (V2.04N, Norcross, Ga.). Scanning electron microscopic(SEM) image analyses were performed (JEOL JSM-5600 instrument, Tokyo,Japan) at 7 kV. High resolution SEM image analyses were performed usinga Focused Ion Beam (FIB/SEM) instrument (Helios 600 Nanolab, FEICompany, Hillsboro, Oreg.) at 20 kV. Particle sizes were measured usinga Beckman Coulter Multisizer 3 analyzer (30-μm aperture, 70,000 counts;Miami, Fla.). The particle diameter (dp) was measured as the 50%cumulative diameter of the volume based particle size distribution. Thewidth of the distribution was measured as the 90% cumulative volumediameter divided by the 10% cumulative volume diameter (denoted 90/10ratio). Viscosity was determined for these materials using a Brookfielddigital viscometer Model DV-II (Middleboro, Mass.). FT-IR spectra wereobtained using a Bruker Optics Tensor 27 (Ettlingen, Germany).Multinuclear (¹³C, ²⁹Si) CP-MAS NMR spectra were obtained using a BrukerInstruments Avance-300 spectrometer (7 mm double broadband probe). Thespinning speed was typically 5.0-6.5 kHz, recycle delay was 5 sec. andthe cross-polarization contact time was 6 msec. Reported ¹³C and ²⁹SiCP-MAS NMR spectral shifts were recorded relative to tetramethylsilaneusing the external standards adamantane (¹³C CP-MAS NMR, δ 38.55) andhexamethylcyclotrisiloxane (²⁹Si CP-MAS NMR, δ-9.62). Populations ofdifferent silicon environments were evaluated by spectral deconvolutionusing DMFit software. [Massiot, D.; Fayon, F.; Capron, M.; King, I.; LeCalvé, S.; Alonso, B.; Durand, J.-O.; Bujoli, B.; Gan, Z.; Hoatson, G.Magn. Reson. Chem. 2002, 40, 70-76]

Example 1

Porous, co-polymeric spherical particles with an average particle sizeof 60 μm are prepared following the process detailed in U.S. Pat. Nos.5,882,521; 5,976,367; 6,106,721; 6,254,780; 6,468,422; and 6,726,842 orU.S. Pat. No. 9,211,524; or 9,120,083 and are surface modified using theprocess detailed in U.S. Pat. Nos. 7,731,844; 8,197,692; or 8,791,220.In particular the surface of the co-polymeric materials arefunctionalized to display a chloromethyl group on the aromaticsubstituent groups

Separately, encapsulating material (<60 μm) are prepared following themethod of Caruso (Phys. Chem. Chem. Phys., 2011, 13, 4782); Sukhishvili(Chem. Mater., 2006, 18 (2), 328); US20150164805; EP2213280; orSchwendeman (J Control Release. 2014; 196:60). Materials used to prepareencapsulating material include (but are not limited to): emulsifiers,materials with varied melting points, materials with different HLB,phospholipids, fatty acids, plant sterols, sorbitan esters, bees wax,carnauba wax, paraffin, stearates, shellac, cellulose derivatives,maltodextrin, starch, gums, cellulose, Polypyrrole, polycarbonate,cetyltrimethylammonium halides, silanes, diblock copolymers, triblockcopolymers such as poly(ethylene oxide)-blockpoly(propyleneoxide)-block-poly(ethylene oxide), named P123 (PEO20PPO70PEO20) and F127(PEO106PPO70-PEO106), alginate, chitosan, xanthan gum, polysaccharide,polysaccharide hydrogel, poly(lysine), poly(acrylic acid), agarose, PEG,poly(hydroxyethylmetacrylate-methyl methacrylate), poly(acrylicacid-co-acrylamide), poly(allylaminehydrochloride),poly(styrenesulfonate sodium salt), poly-(diallyldimethylammoniumchloride), poly(ethylene imine), N-hydroxysuccinimide-PEG,maleimide-PEG-conjugated phospholipids, paraffin, cyclodextrin,carboxymethylated polysaccharide, polycaprolactone, humic substances,Span 60, cholesterol, N-trimethyl chitosan chloride, poly(methylmethacrylate), poly(2-hydroxyethyl methacrylate),poly(N-isopropylacrylamide), poly(N-isopropylmethacrylamide),poly(N-n-propylacrylamide), carboxymethylcellulose, plastic, gold,molecular weight cut-off filters, hybrid organic/inorganic materials,metal oxides, plastics, silica including SBA-15 (PD 50-89 Å) and MCM-41,ceramics, clays, smectic clays, and niosomes.

The encapsulating material contain, or are subsequently modified todisplay an amino-alkyl functional group that is capable of subsequentlyreacting with the chloromethyl groups of the co-polymeric particle,using standard synthetic reactions. There are a number of standardcoupling methods known in the literature, including but not limited toMarch (Advanced Organic Chemistry, 3rd Edition, Wiley, New York, 1985);Odian (The Principles of Polymerization, 2nd Edition, Wiley, New York,1981); and Bioconjugate Techniques (Hermanson, G. T., BioconjugateTechniques; Academic Press: San Diego, 1996))

The encapsulating materials containing peripheral amino-alkyl functionalgroups are then reacted with the chloromethyl groups of the co-polymericparticle to form a covalent attachment between the encapsulatingmaterial and the co-polymeric particle, using standard syntheticreactions. The resulting product comprises a porous, co-polymericparticle with one or more encapsulating materials attached to theperiphery. The linker group between the encapsulating material and theco-polymeric material surface results from the nucleophilic displacementof chlorine to form an alkyl-amino-methyl linker (e.g., {(encapsulatingmaterial)-alkyl-NHCH₂}_(n)(co-polymeric particle) where n is ≥1)

The target molecule is then loaded into the encapsulating materialfollowing methods detailed above. The target molecule includes, but isnot limited to labeling reagents, standards, enzymes (e.g., trypsin,PNGase F, pepsin, IdeS, IdeZ), solution-phase modifiers, reducingagents, alkylating agents, and chloroformate esters. Solution-phasemodifiers include, but are not limited to buffer salts, salts,pH-modifiers such as urea, uric acid, citrate, citric acid, HCl, aceticacid, and reagents that are further modified or decompose to change thesolution phase environment (e.g., urea, carbonates, anhydrides, aceticanhydride).

The resulting target molecule containing encapsulating materials on aporous co-polymeric particle are packaged into a 1 cc sample prep devicecontaining polymeric frits above and below the packing material. Thefrit porosity is selected to be significantly smaller than the productparticle size, as to maintain the sorbent within the device.

The resulting device is used in a sample preparation workflow beforeanalysis using liquid chromatography and mass spectrometry (LC/MS). Thedevice is exposed to solvents and a sample. Since the product is largerthan the frit porosity it is well maintained within the device. Sincethe encapsulating material is attached to the surface of theco-polymeric particle, there is no significant loss of free polymer fromthe device. The advantage of this is the outlet frit does not clog, andhigh permeability is maintained. Also as there is no measurable freepolymer introduced into the LC/MS, there is no significant increase inmatrix effects, enhancement or suppression of signal. Matrix effects,enhancement and suppression are well known in LC/MS.

Because the 60 μm co-polymeric particles prevent loss or leakage ofencapsulating material from the separation device and greatly reducesmatrix effects, suppression or enhancement effects due to theencapsulating material in subsequent LC/MS analysis, this co-polymericparticle is a scaffolding material.

During the workflow the target molecule is released, resulting in thedesired change in workflow. This release of target molecule does notresult in a significant increase in matrix effects, enhancement orsuppression.

Example 2

The process of Example 1 is modified, having the target molecule betrypsin. The workflow consists of the release of trypsin in the presenceof a protein, resulting in the digestion of the protein and formation ofpeptides. Resulting peptides then elute from the device and are furtheranalyzed by LC/MS. Because the trypsin was maintained in theencapsulating material on the scaffolding material it displayed longerstability and good digestion performance. Because the encapsulatingmaterial is immobilized on the scaffolding material there is nosignificant increase in matrix effects, enhancement or suppression.

Example 3

The process of Example 2 is modified, having the target molecule betrypsin. The workflow consists of the release of trypsin in the presenceof a protein, resulting in the digestion of the protein and formation ofpeptides. The formed peptides interact with the porous co-polymericscaffolding material and do not elute from the device until the elutionstrength is suitable (e.g., modification of acetonitrile and watercomposition during an elution step). Resulting peptides then elute fromthe device and are further analyzed by LC/MS. Because the trypsin wasmaintained in the encapsulating material on the scaffolding material itdisplayed longer stability and good digestion performance. Because theencapsulating material is immobilized on the scaffolding material thereis no significant increase in matrix effects, enhancement orsuppression.

Example 4

The process of Example 1-3 is modified to replace the 60 μm co-polymericparticle with a co-polymeric particle preferably that has an averageparticle size in the range of 1-300 μm; more preferably having anaverage particle size in the range of 5-100 μm, more preferably havingan average particle size in the range of 10-70 μm; more preferablyhaving an average particle size in the range of 20-60 μm. The fritporosity is adjusted accordingly to ensure particles are maintainedwithin the sample preparation device.

Example 5

The device detailed in Example 1-4 is modified to a microbore, highpressure column, commonly used in HPLC, UHPLC, or UPLC. Similarworkflows are performed in a constant flow, semi-constant flow,static-pressure (no flow), low pressure (<1,000 psi system pressure) orhigh pressure format (1,000-20,000 system pressure).

Example 6

The scaffolding material of Example 1-5 is modified to include solid,porous solid, non-porous solid, macroporous solid, mesoporous solid,microporous solid, nanoporous solid, superficially porous solid,perfusive solid, controlled pore solid, amorphous solid, radiallyaligned porous solid, non-radially aligned porous solid, circularordered porous solid, crystalline solid, amorphous solid, sinteredsolid, liquid, hydrogel, aerogel, xerogel, cryo-gels, soft-gel, frozen,wax-like, or gel-like material. These scaffolding materials prevent lossor leakage of encapsulating material from the separation device andgreatly reduce matrix effects, suppression or enhancement effects due tothe encapsulating material in subsequent LC/MS analysis.

Example 7

The scaffolding material in Examples 1-6 are modified to includematerials that direct covalent attachment to the internal surface of theseparation device. Exemplary scaffolding materials include, but are notlimited to: wall-anchored monoliths (U.S. Pat. No. 9,289,747 andreferences contained therein), and wall-anchored polymeric high internalphase materials (Silverstein, M. S. and Cameron, N. R. 2010.PolyHIPEs—Porous Polymers from High Internal Phase Emulsions,Encyclopedia of Polymer Science and Technology; Iacono, M., Connolly, D.and Heise, A. Materials 2016, 9, 263; WO 2015/200735; and WO2015/042592). The internal surface of the separation device can include,but are not limited to: steel, stainless steel, titanium, MP35n, PEEK,glass, polymer, polypropylene, polyethylene, copolymers, and Teflon.

The internal surface of the separation device can be modified beforecovalent attachment of the scaffolding material by a number of methodsthat include, but are not limited to: cleaning, washing with solvents,exposure to bases, exposure to acids, plasma pre-treatment, surfaceoxidation with plasma, surface treatment with fluoride sources, surfacetreatment with ozone, surface treatment with nitrogen exposure plasmaassisted chemical vapor deposition, oxygen exposure plasma assistedchemical vapor deposition, air exposure plasma assisted chemical vapordeposition, chemical vapor deposition, molecular vapor deposition,liquid-phase coating approaches, dip-coating, electrochemical coating,initiated polymerization chemical vapor deposition, laser surfacetexturing, remote plasma sputtering. When chemical vapor deposition isemployed the surface can be modified by a number of materials, includingbut not limited to layers of one or more of the following: metals (gold,titanium, silver, iron, nickel, copper, molybdenum, chromium) or oxidesthereof; silanes or siloxanes, silica, or polymers. Preferred silanesresult in surfaces containing silica, organo-silica, or hybridorganic/inorganic materials. Preferred surfaces result from compositionsdetailed in U.S. Pat. Nos. 9,248,383; 9,211,524; 9,145,481; 9,120,083;8,791,220; 8,778,453; 8,697,765; 8,658,277; 7,919,177; 7,223,473;6,686,035; and from compositions detailed in US 20150212056;20150136700; 20150133294; 20140329919; 20140284259; 20140162298;20140096596; 20130319086; 20130206665; 20130135610; 20130112605;20120055860 and 20100076103, all of which are included herein in theirentirety.

Example 8

The process of Example 1-7 is modified to include different types oflinkages between the surface of the encapsulating material and thescaffolding material. The encapsulating material and scaffoldingmaterial can display a variety of different groups that are capable ofreacting using standard synthetic protocols to create a covalentlinkage. There are a number of standard coupling methods known in theliterature, including but not limited to March (Advanced OrganicChemistry, 3rd Edition, Wiley, New York, 1985); Odian (The Principles ofPolymerization, 2nd Edition, Wiley, New York, 1981); and BioconjugateTechniques (Hermanson, G. T., Bioconjugate Techniques; Academic Press:San Diego, 1996)—Repeat.). The resulting product comprises aencapsulated material attached to at least one secondary surface. Thelinker group between the encapsulating material and the scaffoldingmaterial surface is defined in formula I

[(B)—(Y)_(n))]_(o)-EM  (Formula I)

-   -   EM represents the encapsulating material;    -   B represents the scaffolding material;    -   Y is a linker group between the encapsulating material and the        surface of the scaffolding material;    -   o is an integer greater than or equal to 0; and    -   n is an integer greater than or equal to 1.

In particular, Y is a linker group between the encapsulating materialand the co-polymeric material surface, and can be (but not limited to)the formula represented by Formula II

wherein

n¹ an integer from 0-30;

n² aninteger from 0-30;

each occurrence of R¹, R², R³ and R⁴ independently represents hydrogen,fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, aprotected or deprotected alcohol, a zwiterion, or a group Z;

Z represents:

a) a surface attachment group having Formula III:

(B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—   Formula III:

wherein x is an integer from 1-3,

y is an integer from 0-2,

z is an integer from 0-2,

and x+y+z=3

each occurrence of R⁵ and R⁶ independently represents methyl, ethyl,n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted orunsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, aprotected or deprotected alcohol, or a zwiterion group;

B¹ represents —OR⁷, —NR^(7′)R^(7″), —OSO₂CF₃, or —Cl; where each ofR^(7′) R^(7′) and R^(7″) represents hydrogen, methyl, ethyl, n-butyl,iso-butyl, tert-butyl, iso-propyl, thexyl, phenyl, branched alkyl orlower alkyl;

b) an attachment to a surface group through a direct carbon-carbon bondformation or through a heteroatom, ester, ether, thioether, amine,amide, imide, urea, carbonate, carbamate, heterocycle, triazole, orurethane linkage; or

c) an adsorbed, surface group that is not covalently attached to thesurface of the material;

Y′ represents a direct bond; a heteroatom linkage; an ester linkage; anether linkage; an thioether linkage; an amine linkage; an amide linkage;an imide linkage; a urea linkage; a thiourea linkage; a carbonatelinkage; a carbamate linkage; a heterocycle linkage; a triazole linkage;a urethane linkage; a diol linkage; a polyol linkage; an oligomer ofstyrene, ethylene glycol, or propylene glycol; a polymer of styrene,ethylene glycol, or propylene glycol; a carbohydrate group, amulti-antennary carbohydrates, a dendrimer or dendrigraphs, or azwitterion group; and

A represents attachment to the encapsulating material by a ionic group,non-covalently attachment group or by a direct bond including (but notlimited to): a heteroatom linkage; an ester linkage; an ether linkage;an thioether linkage; an amine linkage; an amide linkage; an imidelinkage; a urea linkage; a thiourea linkage; a carbonate linkage; acarbamate linkage; a heterocycle linkage; a triazole linkage; a urethanelinkage; a diol linkage; a polyol linkage; an oligomer of styrene,ethylene glycol, or propylene glycol; a polymer of styrene, ethyleneglycol, or propylene glycol; a carbohydrate group, a multi-antennarycarbohydrates, a dendrimer or dendrigraphs, or a zwitterion group.

Example 9

The product of Examples 1-8 is modified to combine within a separationdevice with a second material of workflow importance. For example, thematerials of example 2-4 having trypsin encapsulated on the surface of aco-polymeric particle is mixed with a secondary particle that hasimmobilized on its surface Protein A. The composition of the Protein Acontaining material includes, but is not limited to: Porous HybridOrganic/Inorganic materials, polymers, sephadex, agarose, cellulose,carbohydrate based, plant based, see-weed based, DVB, PS, DVB/PS,Methacrylate, nonporous and porous. The size of the Protein A containingmaterials can be larger or smaller than the scaffolding material, butneeds to be large enough not to elute through the outlet frit of theseparation device.

In this workflow an antibody isolation on the Protein A resin occurs inthe same separation device as a digestion using the encapsulatedtrypsin. The trypsin is encapsulated during the loading step of theaffinity resin, but is opened during the release of the protein fromProtein A. The trypsin can be optimized for high pH performance, neutralor low pH digestion. Ideally the trypsin is optimized for low pHdigestion which is the same pH used for release of the protein from theaffinity resin. The result of this is low volume release of the affinitycorresponding with digestion in the same well. As detailed in Example 3,when the scaffolding material can allow for retention of the generatedpeptides from the trypsin digestion, an all-in one antibody isolationand digestion device can be realized.

In the case where reduction and alkylation is required, this can beachieved by addition of reduction and alkylation and reduction reagentsbefore or after trypsin digestion. Alternatively the reduction andalkylation reagents can also be encapsulated following the approaches ofExamples 1-8. The resulting separation device contains a number ofencapsulated reagents and enzymes, allowing for a simplified userworkflow. The presence of the scaffolding material prevent loss orleakage of encapsulating material from the separation device and greatlyreduce matrix effects, suppression or enhancement effects due to theencapsulating material in subsequent LC/MS analysis.

Example 10

Example 9 is modified to have an immobilized trypsin sorbent and animmobilized protein A sorbent.

Example 11

Example 9-10 are modified to use a 96 well plate format

Example 12

Examples 9-11 are modified to use in-well plate format and not a flowthrough device. As such no frits are required and larger and smallerScaffolding materials can be used

Example 13

Examples 9-12 are modified to include the use of magnetic particles forthe affinity, digestion, or scaffolding material

Example 14

Examples 9-13 are modified to include other enzymes, including but notlimited to PNGase F, pepsin, IdeS, IdeZ.

Example 15

Examples 9-14 are modified to include other affinity resins, includingbut not limited to: Protein G, Lambda, Kappa, Protein Y, Protein L,aptamers, affimers, amyloids, lectins, or activated resins for usergenerated affinity phases such as streptavidin and epoxy

Example 16

Examples 1-15 are modified to be included in a digestion then affinityworkflow, such as SISCAPA (U.S. Pat. Nos. 9,274,124; 9,261,506;9,170,263; 9,163,276; 9,018,580; 8,916,680; 8,633,031; 8,580,491;8,574,860; 8,569,071; 8,568,988; 8,455,202; 8,187,893; 8,119,356;8,097,425; 7,955,810; and 7,807,172)

Example 17

Examples 1-16 are modified to have a non-covalent interaction betweenthe encapsulating material and the scaffolding material, including butnot limited to: ionic interactions, acid-base interaction, Van de Waalsinteraction, dipole interaction, magnetic attraction, aromaticinteraction and hydrophobic interaction.

Example 18

Porous, co-polymeric spherical particles with an average particle sizeof 60 μm are prepared following the process detailed in Example 1. Tothese materials are adsorbed through a process of solvent evaporation, alow level of trypsin. An encapsulating shell is then formed around theparticle, encapsulating trypsin within the pores of the particle. Thetrypsin is adsorbed to the co-polymeric particle and is not covalentlyattached. The trypsin is protected within the encapsulated shell anddisplays improved storage stability. The trypsin is later released in aprotein digestion workflow, as detailed above.

Example 19

The trypsin adsorbed in the co-polymeric particle having anencapsulating shell, as detailed in Example 18, results in the formationof a non-porous material, as determined by Nitrogen sorption analysis.

Example 20

The trypsin adsorbed in the co-polymeric particle having anencapsulating shell, as detailed in Example 18, results in the formationof a porous material, as determined by Nitrogen sorption analysis.

Example 21

To these porous materials of Example 20 are adsorbed protein alkylationreagents, followed by the formation of a secondary encapsulating shell.This secondary shell has a different release trigger than the primaryencapsulating shell prepared in Example 18. The alkylating reagent isprotected within the encapsulated shell and has no reactivity orinteraction with the trypsin contained in the primary encapsulatedshell, and displays improved storage stability. The alkylation reagentis later released in a protein digestion workflow, as detailed above.

Example 22

To the materials of Example 21 are adsorbed protein reduction reagents,followed by the formation of a tertiary encapsulating shell. Thistertiary shell has a different release trigger than the primary andsecondary encapsulating shells prepared in Example 18 and 21. Thereduction reagent is protected within the encapsulated shell and has noreactivity or interaction with the alkylation reagent or trypsincontained in the secondary or primary encapsulated shells, and displaysimproved storage stability. The reduction reagent is later released in aprotein digestion workflow, as detailed above.

Example 23

The process of example 18-22 is applied to different enzymes andreagents, resulting in the formation of two or more reagentsencapsulated within two or more encapsulating shell layers, within aporous material.

Example 24

Examples 1-23 are modified to be included in a protein deglycosylationworkflow (for example, see the workflows as laid out in Waters'application note: Rapid Preparation of Released N-Glycans for HILICAnalysis Using a Novel Fluorescence and MS-Active Labeling Reagent,Library Number: APNT134829002, Part Number: 720005275EN; Anal. Chem.,2015, 87 (10), pp 5401-5409); Prozyme Application Notes TN 2001 andTN2002)

Example 25

Porous, spherical particles with an average particle size of 60 μm areprepared following the process detailed in Examples 1, 4-9. To thesematerials are adsorbed through a process of solvent evaporation, a lowlevel of PNGase F. An encapsulating shell is then formed around theparticle, encapsulating PNGase F within the pores of the particle. ThePNGase F is adsorbed to the co-polymeric particle and is not covalentlyattached. The PNGase F is protected within the encapsulated shell anddisplays improved storage stability. The PNGase F is later released in aglycoprotein analyis workflow, as detailed above.

Example 26

Porous, spherical particles with an average particle size of 60 μm areprepared following the process detailed in Examples 1, 4-9. To theseparticles are covalently attached reactive groups (reducing agents,alkylating agents, chloroformate esters, acyl chlorides) or proteins,using methods known in the art. The entire system is then encapsulatedby the methods listed above. When ready for use, the encapsulatingmaterial is degraded, swelled, made porous, or otherwise altered to makethe reactive agents or proteins accessible.

Example 27

Examples 1-26 are modified to be included in a workflow for themodification of glyphosate and other highly polar compounds (forexample, see Journal of Chromatography and Separation Techniques Vol 8,Issue 1, Pages 1000346/1-1000346/6)

Example 28

The materials of example 1-26 are used in different devices andworkflows, as detailed in FIGS. 4-10.

Example 29

Porous, spherical particles (the scaffolding material) with an averageparticle size of 60 μm are prepared following the process detailed inExamples 1, 4-9. Onto these materials a reactive fluorescent and MSactive tag (reactive group) such as WATERS TECHNOLOGIES CORPORATIONRAPIFLUOR-MS™, or PROZYME GYKOPREP® INSTANTAB™ or GYKOPREP® INSTANTPC™.An encapsulating shell is then formed around the particle, encapsulatingthe enzyme and/or reactive groups on the surface of, or within the poresof the scaffolding material. A second encapsulation material, containingPNGase F is added around the first. The enzymes and reactive groups areprotected within the encapsulated shell and displays improved storagestability. These groups are later exposed/released for use in theglycoprotein analyis workflow, as detailed above.

Example 30

Porous, spherical particles (the scaffolding material) with an averageparticle size of 60 μm are prepared following the process detailed inExamples 1, 4-9. An encapsulating material containing workflow reagents(proteins, reactive groups, etc) is coated or formed around the surfaceof the scaffolding material. The encapsulating material is thencovalently attached to the surface of the scaffolding material. Whenready for use, the encapsulating material is degraded, swelled, madeporous, or otherwise altered to either release, or make the reactiveagents or proteins accessible.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications, andother references cited herein are hereby expressly incorporated hereinin their entireties by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims.

What is claimed is:
 1. An encapsulated workflow reagent comprising anencapsulating material and a workflow reagent encapsulated within theencapsulating material.
 2. The encapsulated workflow reagent accordingto claim 1, wherein the encapsulating material is attached to a surfaceof a scaffolding material.
 3. The encapsulated workflow reagentaccording to claim 1, wherein the workflow reagent encapsulated withinthe encapsulating material is attached to or adsorbed onto the surfaceof a chromatographic material.
 4. The encapsulated workflow reagentaccording to claim 1, wherein the encapsulating material is one or morepolymers capable of providing a controlled release of the workflowreagents.
 5. The encapsulated workflow reagent according to claim 2,wherein the scaffolding material is a solid, a porous solid, anon-porous solid, a macroporous solid, a mesoporous solid, a microporoussolid, a nanoporous solid, a superficially porous solid, a perfusivesolid, a controlled pore solid, an amorphous solid, a radially alignedporous solid, a non-radially aligned porous solid, a circular orderedporous solid, a crystalline solid, a sintered solid, a liquid, ahydrogel, an aerogel, a xerogel, a cryo-gel, a soft-gel, a gel-likematerial, a wall-anchored monolith, a wall-anchored polymeric highinternal phase material, a particle, a monolith, a membrane, apoly-HIPE, a mesh, a fiber, a screen, an anodized filter, or a frit-likematerial.
 6. The encapsulated workflow reagent according to claim 3,wherein the chromatographic material is a solid, a porous solid, anon-porous solid, a macroporous solid, a mesoporous solid, a microporoussolid, a nanoporous solid, a superficially porous solid, a perfusivesolid, a controlled pore solid, an amorphous solid, a radially alignedporous solid, a non-radially aligned porous solid, a circular orderedporous solid, a crystalline solid, a sintered solid, a hydrogel, anaerogel, a xerogel, a cryo-gel, a soft-gel, a gel-like material, aparticle material, or a monolith material.
 7. The encapsulated workflowreagent according to claim 2, wherein the encapsulated workflow reagentmaterial has the formula[(B)—(Y)_(n))]_(o)-EM  (Formula I) EM represents the encapsulatingmaterial; B represents a scaffolding material; Y is a linker groupbetween the encapsulating material and a surface of the scaffoldingmaterial; o is an integer greater than or equal to 0; and n is aninteger greater than or equal to
 1. 8. The encapsulated workflow reagentaccording to claim 7, wherein the linker group is of the formularepresented by Formula II

wherein n¹ an integer from 0-30; n² an integer from 0-30; eachoccurrence of R¹, R², R³ and R⁴ independently represents hydrogen,fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, aprotected or deprotected alcohol, a zwiterion, or a group Z; Zrepresents: a) a surface attachment group having Formula III:(B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—   Formula III: wherein x is an integer from1-3, y is an integer from 0-2, z is an integer from 0-2, and x+y+z=3each occurrence of R⁵ and R⁶ independently represents methyl, ethyl,n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted orunsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, aprotected or deprotected alcohol, or a zwiterion group; B¹ represents—OR⁷, —NR^(7′)R^(7″), —OSO₂CF₃, or —Cl; where each of R^(7′) R^(7′) andR^(7″) represents hydrogen, methyl, ethyl, n-butyl, iso-butyl,tert-butyl, iso-propyl, thexyl, phenyl, branched alkyl or lower alkyl;b) an attachment to a surface group through a direct carbon-carbon bondformation or through a heteroatom, ester, ether, thioether, amine,amide, imide, urea, carbonate, carbamate, heterocycle, triazole, orurethane linkage; or c) an adsorbed, surface group that is notcovalently attached to the surface of the material; Y′ represents adirect bond; a heteroatom linkage; an ester linkage; an ether linkage;an thioether linkage; an amine linkage; an amide linkage; an imidelinkage; a urea linkage; a thiourea linkage; a carbonate linkage; acarbamate linkage; a heterocycle linkage; a triazole linkage; a urethanelinkage; a diol linkage; a polyol linkage; an oligomer of styrene,ethylene glycol, or propylene glycol; a polymer of styrene, ethyleneglycol, or propylene glycol; a carbohydrate group, a multi-antennarycarbohydrates, a dendrimer or dendrigraphs, or a zwitterion group; and Arepresents attachment to the encapsulating material by a ionic group,non-covalently attachment group or by a direct bond including (but notlimited to): a heteroatom linkage; an ester linkage; an ether linkage;an thioether linkage; an amine linkage; an amide linkage; an imidelinkage; a urea linkage; a thiourea linkage; a carbonate linkage; acarbamate linkage; a heterocycle linkage; a triazole linkage; a urethanelinkage; a diol linkage; a polyol linkage; an oligomer of styrene,ethylene glycol, or propylene glycol; a polymer of styrene, ethyleneglycol, or propylene glycol; a carbohydrate group, a multi-antennarycarbohydrates, a dendrimer or dendrigraphs, or a zwitterion group. 9.The encapsulated workflow reagent according to claim 1, wherein theworkflow reagent is an enzyme, a surfactant, a labeling reagent, areactive compound, an internal standard, an external standard or acombination thereof.
 10. The encapsulated workflow reagent according toclaim 1, wherein the workflow reagent is released over a period of time.11. The encapsulated workflow reagent according to claim 2, wherein theprimary encapsulation shell further encapsulates one or more additionalencapsulation shells comprising one or more independent workflowreagents.
 12. The encapsulated workflow reagent according to claim 11,wherein the one or more additional encapsulation shells are in the formof microcapsules separately contained within the primary encapsulationshell.
 13. The encapsulated workflow reagent according to claim 12,wherein the one or more additional workflow reagents are released at thesame time.
 14. The encapsulated workflow reagent according to claim 12,wherein the one or more additional workflow reagents are releasedsequentially.
 15. The encapsulated workflow reagent according to claim11, wherein the one or more additional encapsulation shells are in theform of concentric encapsulation shells such that the workflow reagentsare sequentially released.
 16. A sample preparation device comprising anencapsulated workflow reagent according to claim
 1. 17. The samplepreparation device according to claim 16, wherein the device is selectedfrom the group consisting of chromatographic columns, thin layer plates,filtration membranes, sample cleanup devices and microtiter plates;packings for HPLC columns; solid phase extraction (SPE); ion-exchangechromatography; magnetic bead applications; affinity chromatographic andSPE sorbents; sequestering reagents; solid supports for combinatorialchemistry; solid supports for oligosaccharide, polypeptides, and/oroligonucleotide synthesis; solid supported biological assays; capillarybiological assay devices for mass spectrometry, templates for controlledlarge pore polymer films; capillary chromatography; electrokinetic pumppacking materials; packing materials for microfluidic devices; polymeradditives; catalysis supports; and packings materials for microchipseparation devices.
 18. A method for a method for preparing a sample foranalysis comprising the steps of: providing a sample preparation devicecomprising an encapsulated workflow reagent according to claim 1;introducing a sample to the sample preparation material; and allowingthe sample to remain with the sample preparation material for sufficienttime to release the encapsulated workflow reagent.
 19. The method for amethod for preparing a sample for analysis according to claim 18 furthercomprising adding a pore forming agent to induce release of theencapsulated workflow reagent.